Magnesium compositions and uses thereof for cognitive function

ABSTRACT

A composition for administration to a subject, such as oral administration to a subject, for example, has been provided. Such a composition may comprise at least one magnesium-counter ion compound. A magnesium-counter ion composition described herein may be useful for any of a variety of applications provided herein, such as maintaining, enhancing, and/or improving health, nutrition, and/or another condition of a subject, and/or cognitive, learning, and/or memory function. A magnesium-counter ion composition provided herein may be useful for administration to a subject presenting magnesium deficiency, mild cognitive impairment, Alzheimer&#39;s disease, attention deficit hyperactivity disorder, ALS, Parkinson&#39;s disease, diabetes, migraine, anxiety disorder, mood disorder, and/or hypertension. A kit, method, and other associated technology are also provided.

BACKGROUND OF THE INVENTION

Magnesium is present in the human body and plays multiple roles. At themolecular level, magnesium is a cofactor for over 300 enzymesresponsible for some of the most important biological activities inmammals, including humans. In living cells, magnesium is involved in thehomeostasis of other minerals, such as sodium, potassium and calcium,and the formation, transfer, storage and utilization of adenosinetriphosphate (ATP), a principal source of energy in living cells. In thehuman body, magnesium is involved in the maintenance of normal muscleand nerve function, heart rhythm, bone strength, and immune systemhealth. Magnesium is also involved in the regulation of blood sugarlevels and the promotion of normal blood pressure.

It has been reported that magnesium plays a role in the regulation ofsynaptic plasticity (Slutsky et al., Neuron, 44, 835-849 (2004)), acellular process believed to be involved in organization of neuralcircuits during early development and in storage of information in laterstages. Magnesium appears to be involved in selective suppression ofso-called background synaptic activity, or background noise, duringwhich meaningful neuronal signals are unaffected. Magnesium thus appearsto increase the signal to noise ratio (S/N) of synaptic transmission andthereby enhance synaptic plasticity.

Synapses are generally less plastic in the aging or diseased brain. Lossof plasticity in the hippocampus, a brain region associated withshort-term memory, may cause forgetfulness that is common in olderpeople. Such loss of plasticity may lead to pathological conditionsassociated with mild cognitive impairment (MCI) or, more seriously, withAlzheimer's disease (AD). As to the latter, it has been reported thatdeceased humans who had been afflicted with AD had significantly lowerlevels of magnesium in regions of their brains than did deceased humansof the same age who had not been afflicted with AD (Andrasi et al.,Magnesium Res. 13(3), 189-196 (2000)). As to aging effects, it has beenreported that supplementing the diet of aging rats with magnesiumappears to increase the expression level of a particular brain molecule,the NMDA receptor, an effect associated with improvement of cognitivefunction (U.S. Patent Application Publication No. US 2006/0089335 A1)

Despite the physiological role of magnesium in human health, people maynot consume enough of the mineral in their diets. Studies have shownthat the dietary intake of magnesium has historically been inadequate inthe U.S. population (Ford et al., (2003) J. Nutr. 133, 2879-2882) orrelatively low for certain population segments (Institute of Medicine,For Calcium, Phosphorus, Magnesium, Vitamin D, and Flouride, 202 and 393(1997)). Magnesium deficit may lead to or may be associated with manypathological symptoms, such as loss of appetite, nausea, vomiting,fatigue, seizures, abnormal heart rhythms, diabetes, and/orcardiovascular disease, for example. According to several studies,magnesium deficit may lead to or may be associated with attentiondeficit hyperactivity disorder (ADHD) in children and symptomsassociated therewith (Kozielec et al., Magnes. Res. 10(2), 143-148(1997) and Mousain-Bosc et al., Magnes. Res. 19(1), 46-52 (2006)).

Commercially available magnesium supplements include magnesium oxidetablets or capsules, various inorganic magnesium compounds, such asmagnesium hydroxide and magnesium sulfate, for example, various organicacid magnesium salt compounds, such as magnesium salts of gluconic acid,citric acid, and lactic acid, for example, and various magnesium chelatecompounds. Magnesium oxide may be high in elemental magnesium content,but very low in magnesium bioavailability, or absorption rate in thehuman body (Ranade et al., Am. J. Therapeut. 8(5), 345-357 (2001)).Inorganic magnesium compounds, such as magnesium hydroxide and magnesiumsulfate, may also be poor in terms of magnesium bioavailability and maygive rise to an undesirable side-effect, diarrhea. Organic acidmagnesium salt compounds, such as magnesium salts of gluconic acid,citric acid and lactic acid, may be associated with gastrointestinaldistress, laxative effect, and/or diarrhea. While various so-calledmagnesium chelate compounds have been promoted as having bettermagnesium bioavailability, these compounds may be highly alkaline andpoor in terms of palatability.

The recommended daily intake of magnesium for an adult is generally fromabout 15 mmol to 20 mmol (30 mEq to 40 mEq), and normal magnesium serumlevels range from 0.7 mmol/L to 1.0 mmol/L. Foods that are rich inmagnesium include legumes, whole grains, green leafy vegetables, nuts,coffee, chocolate and milk. Although these foods are readily available,some individuals do not consume adequate quantities to satisfy the dailynutritional requirement. Furthermore, expanded consumption of processedfoods, which tend to contain less magnesium, may account for theperceptible decline in dietary magnesium in the United States during thepast century. Thus, continued use of an oral magnesium supplement thatoffers reliable absorption and bioavailability is recommended for peoplewith magnesium deficiency. Oral magnesium supplements are available in anumber of formulations that utilize a different anion or salt—such asoxide, gluconate, chloride or lactate dihydrate. However, thesepreparations are not interchangeable because they have differences inabsorption, bioavailability and palatability.

Magnesium is absorbed primarily in the distal small intestine, andhealthy people absorb approximately 30% to 40% of ingested magnesium.Since magnesium is predominately an intracellular cation, theeffectiveness of a dosage form is assessed by its solubility and rate ofuptake from the small intestine into the bloodstream and by its transferinto the tissues. Magnesium balance is regulated by the kidneys. Whenmagnesium levels in the blood are high, the kidneys will rapidly excretethe surplus. When magnesium intake is low, on the other hand, renalexcretion drops to 0.5 mmol to 1 mmol (1 mEq to 2 mEq) per day.

Means for providing magnesium to the human body as a supplement havebeen proposed in the art. For example, for the treatment of arrhythmia,magnesium sulfate has been intravenously administered to patients. Otherdietary supplements have included magnesium oxide, magnesium hydroxideand magnesium carbonate. Despite the ability of these compounds toincrease magnesium levels, they are primarily insoluble in thegastrointestinal tract, and hence, not easily delivered to thegastrointestinal system, without side-effects. As such, there is aconsiderable need for improved magnesium compositions, uses thereof,and/or associated technology. The subject invention satisfies theseneeds and provides related advantages as well.

SUMMARY OF THE INVENTION

A composition for administration to a subject is described herein. Sucha composition may comprise at least one magnesium-comprising component(MCC) or also used herein as magnesium-counter ion compound. Examples ofan MCC include a magnesium salt of an amino acid, magnesium acetate,magnesium ascorbate, magnesium citrate, magnesium gluconate, magnesiumlactate, magnesium malate, magnesium pyrrolidone carboxylate, magnesiumtaurate, and magnesium threonate. Such a composition may comprise atleast one component of non-acidified milk sufficient to enhancebioavailability of elemental magnesium associated with the MCC. Examplesof such a component include lactose, a fatty acid or milk fat, and/oranother organic component thereof, for example, sufficient for suchenhancement. A mass ratio of the amount of elemental magnesiumassociated with the at least one MCC and the amount of the component maybe from about 1 to about 5 to about 1 to about 3000. Such a compositionmay be suitable for oral administration to a subject.

In one embodiment, the present invention provides an oral dosage formcomprising 300 mg to 1.5 g of magnesium threonate. The oral dosage formcan be a tablet, formulated in form of liquid, in immediate or sustainedrelease format. In some aspects, the oral dosage form comprises aplurality of beads encapsulated in a capsule. Such format can be used asa sustained release formulation.

In another embodiment, the present invention provides amagnesium-containing composition that has the following characteristics:(a) the magnesium contained therein has a weight percentage of at leastabout 8%; (b) a counter ion comprises at least two hydroxyl groups; (c)the composition has a solubility of at least at least 20 mg/mL; and (d)the composition exhibit a pH value between about 6 to 8.5 when dissolvedin water.

The present invention also provides a magnesium-containing an oraldosage that comprises a pharmaceutically active agent and an excipient,wherein the excipient is magnesium thereonate

Further provided in the present invention is a food compositioncomprising a food carrier and a magnesium-containing compound where themagnesium-containing compound is characterized in that: a) the carboncontained therein has a weight percentage of at least about 8% of theweight of a counter ion; b) a counter ion comprises at least twohydroxyl groups; c) the composition has a solubility of at least about20 mg/mL; and d) the composition exhibits a pH value between about 6-8.5when dissolved in water. In some embodiments, the magnesium containingcompound comprises magnesium threonate. In other embodiments, the foodcomposition is packaged as a beverage, a solid food or a semi-solidfood. In still other embodiments the food composition is packaged as asnack bar, a cereal product, a bakery product or a dairy product. Thefood composition may be milk or a soft drink. In some embodiments, thefood composition comprises: an effective amount of magnesium or saltthereof for modulating cognitive function in a subject in need thereof;and a food carrier. Where desired, the food composition comprisesmagnesium threonate. In some embodiments, the food composition containsmagnesium or a salt thereof present in an amount effective to enhanceshort-term memory or long-term memory, ameliorate dementia or amelioratedepression. Also provided is a food supplement comprising magnesiumthreonate. Also provided is a method of preparing a food supplementcomprising mixing magnesium threonate with a food additive agent. Insome embodiments, the food additive agent is a sweetening agent, aflavoring agent, a coloring agent, a filling agent, a binding agent, alubricating agent or a preservative agent.

A composition, kit, and/or a method described herein may be useful forpurposes described herein, such as maintaining, enhancing, and/orimproving health, nutrition, and/or another condition of a subject,and/or cognitive, learning, and/or memory function, for example, such asmagnesium deficiency, mild cognitive impairment (MCI), Alzheimer'sdisease (AD), attention deficit hyperactivity disorder (ADHD),amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, Parkinson'sdisease, diabetes, migraine, anxiety, mood, and hypertension, merely byway of example.

A method of providing magnesium supplementation to a subject isdescribed herein. Such a method may comprise administering to thesubject at least one MCC, such as any of those described above. Such amethod may comprise administering to the subject at least one componentof non-acidified milk sufficient to enhance bioavailability of elementalmagnesium associated with the MCC, such as any of those described above.A mass ratio of the amount of elemental magnesium associated with the atleast one MCC and the amount of the component maybe as described above.Such a method may comprise oral administration to the subject.

In one embodiment, the present invention provides a method of enhancingcognitive function. The method comprises administering to a subject anamount of magnesium-containing compound effective to achieve aphysiological concentration of magnesium at about 0.75 mM or above,wherein said concentration of magnesium is measured under a fastingcondition. In some instances, the concentration of magnesium is measuredafter fasting for at least about twelve hours. In other instances, thephysiological concentration is serum concentration, plasmaconcentration, or cerebrospinal fluid concentration. In someembodiments, the magnesium-containing compound is a magnesium-counterion compound. In other embodiments, the counter ion is an organic ion.In other instances the organic counter ion is threonate. In someembodiments, the magnesium-containing compound is amagnesium-supplemented foodstuff. Also provided is a method where thecognitive function is short-term memory or long-term memory. In someinstances, the physiological concentration is maintained for a period ofgreater than one month.

In one embodiment, a method of maintaining cognitive function isprovided wherein the method comprises administering to a subject anamount of magnesium-containing compound effective to increase aphysiological concentration of magnesium by at least about 10% ascompared to an initial level of magnesium prior to the administration.In some instances the increase is measured under a fasting condition. Inother instances, the physiological concentration is serum concentration,plasma concentration, or cerebrospinal fluid concentration. In someembodiments the magnesium-containing compound is a magnesium-counter ioncompound. In other embodiments the counter ion is an organic counterion. In a particular embodiment the organic counter ion is threonate. Insome embodiments, the magnesium-containing compound is amagnesium-supplemented foodstuff. In still further embodiments, theconcentration is maintained for a period of greater than four months. Inyet another embodiment, the method comprises the step of determiningstarting physiological magnesium concentration of the subject under afasting condition.

Also provided is a method of maintaining and/or enhancing cognitivefunction comprising administering to a subject an amount ofmetal-organic counter ion complex effective to increase a physiologicalconcentration of threonate by at least about 10% as compared to aninitial level of threonate prior to said administration. In someinstances the metal-organic counter ion complex comprises threonate as acounter ion.

In another aspect of the invention a method for therapeutic orprophylactic treatment of a cognitive dysfunction is provided, whereinthe method comprises administering to a subject in need of therapeuticor prophylactic treatment of cognitive dysfunction amagnesium-containing composition to yield a level of physiologicalconcentration of magnesium sustained at the level of 0.75 mM or abovefor at least about 15 days. In some instances, the magnesium issustained at the level of 0.75 mM or above for at least about one monthor at least about four months. In other instances, magnesiumconcentration is magnesium plasma concentration measured after fastingfor at least about eight hours. In some embodiments, the subject is anadult. In other embodiments, the subject is a patient suffering from ordiagnosed with dementia or Alzheimer's disease.

Where desired, one can administer to a subject an amount ofmagnesium-containing compound effective to achieve a physiologicalconcentration of magnesium at about 0.78 mM, 0.8 mM, 0.82 mM, 0.84 mM,0.86 mM, 0.88 mM, 0.90 mM, 0.92 mM, 0.94 mM, 0.96 mM, 0.98 mM, or above.In one aspect, such magnesium concentration is maintained for at least 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5years, 2 years, or even longer. Preferably, the concentration ofmagnesium is measured under a fasting condition, e.g., after fasting forat least about 8 hours, 10 hours, 12 hours, 15 hours, 24 hours, or evenlonger. The physiological concentration of magnesium can be serumconcentration, plasma concentration, or cerebrospinal fluidconcentration. Such physiological concentration can be determined bymeasuring intracellular ionized magnesium in red blood cells, bonemagnesium content, magnesium concentration in the cerebrospinal fluid, asublingual magnesium assay intracellular free magnesium, or nuclearmagnetic resonance spectroscopy. In some aspect, themagnesium-containing compound is effective in improving short-term orlong-term memory.

In a related embodiment, the present invention provides a method oftherapeutic or prophylactic treatment of cognitive dysfunction,comprising: administering to a subject in need for a therapeutic orprophylactic treatment of cognitive dysfunction a composition ofmagnesium that yields a sustained level physiological concentration ofmagnesium of 0.75 mM or above for at least about 15 days, e.g. upon,multiple dosages. Preferably, the beneficial effect can last longer than20 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 1 year, 1.5 years, 2 years or longer.

In another embodiment, the present invention provides a method ofameliorating the effects of a neurological disorder. The methodcomprises administering to a subject an amount of magnesium-containingcompound effective to increase a physiological concentration ofmagnesium by at least about 10% as compared to an initial level ofmagnesium prior to the administration. In some instances, the increaseis measured under a fasting condition. In other instances theconcentration of magnesium is measured after fasting for at least abouttwelve hours. In some embodiments of this method, the neurologicaldisorder is dementia, Alzheimer's disease or depression. In otherembodiments of the method, the physiological concentration is serumconcentration, plasma concentration or cerebrospinal fluidconcentration. In some embodiments of this method, themagnesium-containing compound is a magnesium-counter ion compound. Wheredesired, the counter ion is an organic ion. In a particular embodiment,the organic counter ion is threonate. In some instances, themagnesium-containing compound is a magnesium-supplemented foodstuff. Insome instances of this method, the concentration is maintained for aperiod of greater than four months. In other embodiments, the methodfurther comprises the step of determining starting physiologicalmagnesium concentration of the subject under a fasting condition.

Yet another aspect of the present invention provides a method oftherapeutic or prophylactic treatment of a neurological disorder,comprising administering to a subject in need of therapeutic orprophylactic treatment of said neurological disorder, amagnesium-containing composition to yield a sustained level ofphysiological concentration of magnesium of 0.75 mM or above for atleast about 15 days. In some embodiments, the composition of magnesiumyields a sustained level of physiological concentration of magnesium of0.75 mM or above for at least about one month or at least about fourmonths. In some instances, the neurological disorder is dementia,depression or Alzheimer's disease.

In still another embodiment, a method of therapeutic or prophylactictreatment of a neurological disorder is provided where the methodcomprises comprising administering to a subject an amount ofmetal-organic counter ion complex effective to increase a physiologicalconcentration of threonate by at least about 10% as compared to aninitial level of threonate prior to said administration. In someinstances, the metal-organic counter ion complex comprises threonate asa counter ion.

Also provided is a method of ameliorating the effects of a metabolicdisorder comprising administering to a subject an amount ofmagnesium-containing compound effective to increase a physiologicalconcentration of magnesium by at least about 10% as compared to aninitial level of magnesium prior to said administration. In someinstances the concentration of magnesium is measured after fasting forat least about twelve hours. In other instances, the physiologicalconcentration is serum concentration, plasma concentration, orcerebrospinal fluid concentration. In some embodiments of this methodthe magnesium-containing compound is a magnesium-counter ion compound.In other embodiments, the counter ion is an organic ion. In a particularembodiment, the organic counter ion is threonate. In some embodiments,the magnesium-containing compound is a magnesium-supplemented foodstuff.In some embodiments, the metabolic disorder is diabetes. In otherembodiments, the concentration is maintained for a period of greaterthan 1 month.

In still another aspect of the present invention a method of therapeuticor prophylactic treatment of a metabolic disorder is provided, whereinthe method comprises administering to a subject in need of therapeuticor prophylactic treatment of a metabolic disorder a magnesium-containingcomposition to yield a level of physiological concentration of magnesiumsustained at the level of 0.75 mM or above for at least about 15 days.In some instances, the magnesium is sustained at the level of 0.75 mM orabove for at least about 1 month or at least about four months. In otherinstances, magnesium concentration is magnesium plasma concentrationmeasured after fasting for at least about 8 hours. In some embodiments,the subject is an adult.

In yet another aspect of the present invention, a method of therapeuticor prophylactic treatment of a metabolic disorder is provided comprisingadministering to a subject an amount of metal-organic counter ioncomplex effective to increase a physiological concentration of threonateby at least about 10% as compared to an initial level of threonate priorto said administration. In some embodiments the metal-organic counterion complex comprises threonate as a counter-ion. In other embodiments,the metal-organic counter ion complex is magnesium threonate. In stillother embodiments, the metal-organic counter ion complex is administeredorally. In still other embodiments, the metal-organic counter ioncomplex is provided as a food supplement.

Another embodiment provides a method of extending lifespan of a subjectcomprising administering to said subject an amount ofmagnesium-containing compound effective to achieve a physiologicalconcentration of magnesium of about 0.75 mM or above, thereby extendingthe lifespan of said subject, wherein said concentration is measuredunder a fasting condition. In some embodiments, the concentration ofmagnesium is measured after fasting for at least about twelve hours. Inother embodiments, the physiological concentration is serumconcentration, plasma concentration, or cerebrospinal fluidconcentration. In some embodiments, the magnesium-containing compound isa magnesium-counter ion compound. In other embodiments, the counter ionis an organic counter ion. In a particular embodiment, the organiccounter ion is threonate. In some embodiments, the saidmagnesium-containing compound is a magnesium-supplemented foodstuff. Insome embodiments, the concentration is maintained for a period ofgreater than 1 month.

Another embodiment provides a method of extending lifespan of a subjectcomprising administering to a subject an amount of magnesium-containingcompound effective to increase a physiological concentration ofmagnesium by at least about 10% as compared to an initial level ofmagnesium prior to said administration. In some embodiments, theincrease is measured under a fasting condition. In some embodiments, thephysiological concentration is serum concentration, plasmaconcentration, or cerebrospinal fluid concentration. In someembodiments, the magnesium-containing compound is a magnesium-counterion compound. In some embodiments, the counter ion is an organic counterion. In some embodiments, the organic counter ion is threonate. In someembodiments, the magnesium-containing compound is amagnesium-supplemented foodstuff. In some embodiments, the concentrationis maintained for a period of greater than 4 months. In someembodiments, the method further comprises the step of determiningstarting physiological magnesium concentration of said subject under afasting condition.

Still another embodiment of the present invention provides a method ofextending lifespan of a subject comprising administering to a subject anamount of metal-organic counter ion complex effective to increase aphysiological concentration of threonate by at least about 10% ascompared to an initial level of threonate prior to said administration.In some embodiments, the metal-organic counter ion complex comprisesthreonate as a counter-ion.

Also provided is a method of determining an effective amount ofmagnesium to produce a physiological effect, comprising the steps of: a)obtaining a sample from a subject being tested, wherein said sample istaken under a fasting condition; b) determining a physiologicalconcentration of magnesium from said sample; and c) providing thesubject with a magnesium-containing compound dosing regimen effective toachieve a physiological concentration of magnesium of about 0.75 mM orabove. In some embodiments, the concentration of magnesium is measuredafter fasting for at least about twelve hours. In other embodiments, thephysiological concentration is serum concentration, plasmaconcentration, or cerebrospinal fluid concentration. In someembodiments, the magnesium-containing compound is a magnesium-counterion compound. In still other embodiments, the counter ion is an organiccounter ion. In a particular embodiment, the organic counter ion isthreonate. In some embodiments, the magnesium-containing compound is amagnesium-supplemented foodstuff. In another embodiment, the methodfurther comprises the step of determining a physiological concentrationof magnesium after said subject has begun said dosing regimen.

Another embodiment of the present invention provides a method ofdetermining an effective amount of magnesium to produce a physiologicaleffect, comprising the steps of: a) obtaining a sample from a subjectbeing tested, wherein said sample is taken under a fasting condition; b)determining a physiological concentration of magnesium from said sample;and c) providing said subject with a magnesium-containing compounddosing regimen effective to achieve an increase in a physiologicalconcentration of magnesium by at least about 10% as compared to aninitial level of magnesium measured under a fasting condition.

Where desired, the amount of magnesium-containing compound is effectiveto increase a physiological concentration of magnesium by at least about12%, 14%, 15%, 20%, 25% or more as compared to an initial level ofmagnesium prior to said administration. The increase in physiologicalconcentration of magnesium can last for at least 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2 years, oreven longer. As noted herein, the increase in physiologicalconcentration of magnesium is preferably measured after a fastingcondition. The neurological disorders that can be ameliorated by thesubject method include but are not limited to dementia, Alzheimer'sdisease, and depression. In a related but separate embodiment, thepresent invention provides a method of ameliorating depression byadministering to a subject in need for a therapeutic or prophylactictreatment of depression, a composition of magnesium to yield a sustainedlevel of physiological concentration of magnesium of 0.75 mM or abovefor at least about 15 days, e.g. upon multiple dosages. Preferably, thebeneficial effect can last longer than 20 days, 25 days, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2years or longer.

In yet another embodiment, the present invention provides a method ofincreasing bone density. The method comprises the step of administeringto a subject in need for a therapeutic or prophylactic treatment of bonedensity a composition of magnesium to be sustained at the level of 0.75mM or above for at least about 15 days, 20 days, 25 days, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2years or longer.

In still another embodiment, the present invention provides a method ofextending lifespan of a subject comprising administering to said subjectan amount of magnesium-containing compound effective to achieve aphysiological concentration of magnesium of about 0.75 mM or above,thereby extending the lifespan of said subject, wherein saidconcentration is measured under a fasting condition. Also provided in arelated embodiment is a method of increasing expected life span of asubject, comprising: administering to a subject a composition ofmagnesium to yield a sustained level of physiological concentration ofmagnesium of 0.75 mM or above for at least about 15 days, 20 days, 25days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year,1.5 years, 2 years or longer.

The present invention also provides a method of determining an effectiveamount of magnesium to produce a physiological effect. The methodcomprises the steps of (a) obtaining a sample from a subject beingtested, wherein said sample is taken under a fasting condition; (b)determining a physiological concentration of magnesium from said sample;and (c) providing said subject with a magnesium-containing compounddosing regimen effective to achieve a physiological concentration ofmagnesium of about 0.75 mM or above. In a related but separateembodiment, the method of determining an effective amount of magnesiumto produce a physiological effect comprises the steps of (a) obtaining asample from a subject being tested, wherein said sample is taken under afasting condition; (b) determining a physiological concentration ofmagnesium from said sample; and (c) providing said subject with amagnesium-containing compound dosing regimen effective to achieve anincrease in a physiological concentration of magnesium by at least about10% as compared to an initial level of magnesium measured under afasting condition. The physiological effect encompasses enhancedcognitive function (e.g., short-term memory or long-term memory),ameliorating an effect of a neurological disorder such as Alzheimer'sdisease or depression.

These and various other aspects, features, and embodiments are furtherdescribed herein. Any other portion of this application is incorporatedby reference in this summary to the extent same may facilitate a summaryof subject matter described herein, such as subject matter appearing inany claim or claims that may be associated with this application.

In a related but separate embodiment, the present invention provides anoral dosage form comprising about 0.1 mg to 800 mg of magnesiumthreonate. Where desired the oral dosage form comprises between about 1mg to about 100 mg, 10 mg to about 500 mg, or more magnesium threonate.In some embodiment, the oral dosage form is substantially free ofexcipient. The oral dosage form can be in form of a tablet, capsule, orany other known format. The present invention also provides foodsupplements comprising the subject MCC or magnesium-counter ioncompound.

Also provided is a method of determining an amount ofmagnesium-containing component that is needed to produce a physiologicaleffect in a subject, comprising the steps of:

-   -   a. obtaining a sample of biological fluid from the subject; and    -   b. calculating the amount of magnesium to be supplied to said        subject according to the formula of:

Mg _(x) =GFR·T·Mg _(mw) ·k _(e)·([Mg] _(o) ² −[Mg] _(o) ¹)/k _(x)

-   -   -   wherein Mg_(x) is effective amount of magnesium to be            supplied to said subject;        -   wherein [Mg]₀ ¹ is the initial concentration of magnesium in            extracellular compartment;        -   wherein K_(x) is bioavailability of said            magnesium-containing component;        -   wherein GFR is glomerular filtration rate;        -   wherein k_(e) is the excretion rate of filtered Mg in            kidney;        -   wherein T is time in hours;        -   wherein Mg_(mw) is molecular weight of the element            magnesium; and        -   wherein [Mg]₀ ² is a desired concentration of magnesium to            be achieved upon supplementing said subject the determined            amount of magnesium-containing component.

In some embodiments, the concentration of magnesium in said biologicalfluid is measured under a fasting condition. In some embodiments, theconcentration of magnesium is measured after fasting for at least abouttwelve hours. In some embodiments, the biological fluid is selected fromblood, serum and, plasma. In some embodiments, the amount of magnesiumsupplied is effective to achieve an increase in a physiologicalconcentration of magnesium by at least about 5% as compared to aninitial level of magnesium measured under a fasting condition.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

A description of various aspects, features, embodiments, and examples isprovided herein with reference to the accompanying drawings, which arebriefly described below. The drawings may illustrate one or moreaspect(s), feature(s), embodiment(s), and/or example(s) in whole or inpart. The drawings are illustrative and are not necessarily drawn toscale.

FIG. 1 is a graphical presentation of results of a taste test concerningtwo different compositions comprising milk and various sources ofmagnesium as further described in Example 2.

FIG. 2 is a graphical presentation of the enhancement of the magnesiumabsorption rate in four groups of young adult rats that were exposed,respectively, to four different compositions: 1) magnesium gluconate (12mM) in skim milk; 2) magnesium gluconate (12 mM) in milk prepared frompowdered milk; 3) magnesium gluconate (12 mM) in water comprising 1%cream; or 4) magnesium gluconate (12 mM) in water comprising 5 weightpercent lactose. The enhancement of the magnesium absorption wasmeasured as a percentage relative to the magnesium absorption rate in acontrol group of young adult rats that were exposed to a compositioncomprising magnesium gluconate (12 mM) and water, as further describedin Example 3.

FIG. 3 is a graphical presentation of the magnesium absorption rate inyoung adult rats that were exposed to a composition of a mixture ofmagnesium-counter ion components and water and the magnesium absorptionrate in young adult rats that were exposed to a composition of the samemixture of magnesium-counter ion components and skim milk, as furtherdescribed in Example 4.

FIG. 4 is a graphical presentation of the magnesium absorption rate inyoung adult rats that were exposed to a composition of magnesiumchloride and water, magnesium gluconate and skim milk, or magnesiumgluconate and in water comprising 5 weight percent lactose, versus theelemental magnesium intake (mg/day/rat), as further described in Example5.

FIG. 5 is a graphical presentation of the magnesium absorption rate inyoung adult rats that were exposed to a composition of magnesiumchloride and water, or magnesium threonate and water, versus theelemental magnesium intake (mg/day/rat), as further described in Example6.

FIG. 6 is a graphical presentation of the average concentration ofmagnesium in serum taken from young adult rats that were exposed to acomposition of magnesium chloride and water, magnesium threonate andwater, or a mixture of magnesium gluconate, magnesium lactate, magnesiumcitrate and skim milk, or de-ionized water, as further described inExample 7.

FIG. 7 is a graphical representation of the average percentageimprovement of spatial working memory results for various young and agedrats that were fed various diets, plotted for various days of a trainingand testing period (panels A and B); and the percentage enhancement inyoung and aged rats receiving magnesium supplementation (panel C).

FIG. 8 is a graphical representation of experimental data showing therestorative effect of magnesium on short-term recognition memory inrats. The top portion of the figure is a graphical representation of theexperimental methodology.

FIG. 9 is a graphical representation of experimental data showing theincrease in the time course of recognition memory decline in rats givenmagnesium. The top portion of the figure is a graphical representationof the experimental methodology.

FIG. 10 is a graphical representation of results from an elevated T-mazetask for young and old rats. The represented data demonstrate thatmagnesium improves working and short-term spatial memory in aging rats.The top portion of the figure is a graphical representation of theexperimental methodology.

FIG. 11 is a graphical representation of experimental resultsenhancement of short term memory in rats receiving a magnesium mixtureand 5% lactose.

FIG. 12 is a graphical representation of experimental results from awater maze test conducted on young and aged rats. The represented datashow that magnesium threonate supplementation leads to enhancement oflearning and long-term memory in both young and aged rats.

FIG. 13 is a graphical representation of the results of a memory testconducted on young and aged rats. The data demonstrates that magnesiumsupplementation enhance memory in both populations.

FIG. 14 is a graphical representation of experimental results frompattern completion tests conducted on aged rats. The data demonstratesthe effects of magnesium threonate on the memory process. The topportion of the figure is a graphical representation of the experimentalmethodology.

FIG. 15 is a graphical representation of the effects of magnesiumthreonate on the memory process in a mouse model of Alzheimer's Disease(AD). The data demonstrates that both learning (panels A and C) andmemory (panels B and D) at both 6 and 13 months are improved when ADmice are given magnesium threonate.

FIG. 16 is a graphical representation of the results from a learning(panel A) and memory (panel B) comparison of magnesium threonatetreatment with drugs aricept or memantine used to treat AD.

FIG. 17 is a graphical representation of serum concentration levels ofmagnesium in men and women.

FIG. 18 is a graphical representation of serum concentration levels ofmagnesium in women between the ages of 18 and 35.

FIG. 19 is a graphical representation of the correlation of magnesiumintake and short-term memory effects.

FIG. 20 is a graphical representation of the correlation of plasmaconcentration of magnesium and short-term memory effects.

FIG. 21 is a graphical representation of the correlation betweenmagnesium intake and increased motility in mice with and without AD atboth 7 months and 15 months.

FIG. 22 is a graphical representation of the antidepressant effects ofmagnesium.

FIG. 23 is a graphical representation of the effect of magnesium on thelifespan of Drosophila.

FIG. 24 is a graphical representation of the correlation betweenlifespan increase and magnesium intake in Drosophila.

FIG. 25 is a graphical representation of the bioavailability ofdifferent magnesium-containing compositions.

FIG. 26 is a graphical representation of the correlation betweenmagnesium concentration in the brain, the amount of magnesium intake(panel A) and the correlation between short term memory effects (panelB).

FIG. 27 is a graphic representation of the effectiveness of magnesiumthreonate, compared with magnesium gluconate in milk, in absorption bythe brain (panel A). Also shown is a comparison of the results of amemory test using magnesium threonate (panel B) and magnesiumgluconate+milk (panel C).

FIG. 28 is a graphic representation of a method of determining aneffective magnesium dosing regimen based on basal magnesiumconcentration under fasting conditions. Panel A demonstrates therelationship between blood and urine magnesium concentration and Panel Bshows the use of magnesium concentration in the extracellularcompartment and in urine to determine proper dosing.

FIG. 29 shows the protection of synapse loss in AD mice by magnesiumthreonate treatment. Panel A demonstrates the lower synapses count indentate gyrus of hippocampus of AD mice. Panel B demonstrates the highersynaptic density in the same region. Panel C demonstrates thequantitative comparison of the synaptic densities in AD mice, AD micewith MgT treatment, and wild type mice.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

It will be understood that a word appearing herein in the singularencompasses its plural counterpart, and a word appearing herein in theplural encompasses its singular counterpart, unless implicitly orexplicitly understood or stated otherwise. Further, it will beunderstood that for any given component described herein, any of thepossible candidates or alternatives listed for that component, maygenerally be used individually or in any combination with one another,unless implicitly or explicitly understood or stated otherwise.Additionally, it will be understood that any list of such candidates oralternatives, is merely illustrative, not limiting, unless implicitly orexplicitly understood or stated otherwise. Still further, it will beunderstood that any figure or number or amount presented herein isapproximate, and that any numerical range includes the minimum numberand the maximum number defining the range, whether the word “inclusive”or the like is employed or not, unless implicitly or explicitlyunderstood or stated otherwise. Generally, the term “approximately” or“about” or the symbol “˜” in reference to a figure or number or amountincludes numbers that fall within a range of ±5% of same, unlessimplicitly or explicitly understood or stated otherwise. Yet further, itwill be understood that any heading employed is by way of convenience,not by way of limitation. Additionally, it will be understood that anypermissive, open, or open-ended language encompasses any relativelypermissive to restrictive language, less open to closed language, orless open-ended to closed-ended language, respectively, unlessimplicitly or explicitly understood or stated otherwise. Merely by wayof example, the word “comprising” may encompass “comprising”-,“consisting essentially of”-, and/or “consisting of”-type language.

A magnesium-counter ion composition, a kit, and/or a method describedherein may be useful for purposes described herein, such as maintaining,enhancing, and/or improving health, nutrition, and/or another conditionof a subject, and/or cognitive, learning, and/or memory function, forexample, such as magnesium deficiency, mild cognitive impairment (MCI),Alzheimer's disease (AD), attention deficit hyperactivity disorder(ADHD), ALS, Parkinson's disease, diabetes, migraine, anxiety, mood, andhypertension, merely by way of example. A description of variousaspects, features, embodiments, and examples, is provided herein.

The body magnesium level among human population varies from person toperson, approximately distributed according to a Gausian curve. Forexample, in a survey among 9506 white males and females the serum Mglevels were distributed between about 0.75 mM and about 0.95 mM withmost subjects having a serum magnesium level near the middle of thedistribution. The distribution in men and women is shown in FIG. 17(adopted from Kao et al., Arch. Intern. Med. 159: 2151-9 (1999); FIG.18). The distribution in serum magnesium levels among young and healthywomen has also been reported and show a similar distribution pattern, asshown in FIG. 18 (adopted from Cole and Quamme, J. Amer. Soc. Nephrol.11: 193747 (2000)). However, other studies have shown that blood (serumor plasma) magnesium levels in AD patients are approximately 20% lowerthan healthy control groups. See, e.g., Lemke, Biol. Psychiatry. 37:341-3 (1995); Cilliler et al. Gerontology. 53: 419-22 (2007).

A number of methods have been used to assess the body magnesium levelsin humans. These methods differ from one another in the type of sampleand the analytical technique used. Serum and plasma have been the twomost commonly used types of samples although some studies used red bloodcells or tissue samples. Among the Mg detection techniques used are:absorbance-based dye technique, atomic absorption technique,ion-selective electrode technique and NMR technique. The first twotechniques measure the total magnesium concentration, which include bothionized free Mg²⁺ and Mg²⁺ bound to proteins and other molecules in thesample, while the latter two techniques measure only ionized magnesium.

A major problem with the various methods mentioned above is the lack ofa standardized test including a standardized condition under which atest is performed. There is also poor understanding about theinterrelation between the experimental values obtained from the variousmethods. For this reason, the range of blood magnesium (serum or plasma)levels reported for healthy subjects or patients vary widely from studyto study and from lab to lab. For example, Cilliler, et al. reportedthat the average serum Mg levels for AD patients diagnosed as mild andmoderate, AD patients diagnosed as severe, and non-AD control subjectswere 0.92 mM (2.197 mg/dl), 0.88 mM (2.11 mg/dl) and 1.05 mM (2.51mg/dl), respectively. Although the trend for blood magnesium levelbetween AD patients and their healthy control subjects is consistentwith earlier findings, the absolute values of the serum magnesium levelsdetermined by these authors are significantly higher than those reportedelsewhere. For example, the 0.92 and 0.88 mM serum magnesiumconcentrations reported by Cilliler, et al. are even higher than themeans of serum magnesium concentration for healthy people shown in FIGS.17 and 18. In another study by Garba, et al. the average serum Mg levelamong 20 healthy subjects aged from 18 to 40 was only 0.27 mM (640μg/dl).

Further contributing to the confusion is the lack of a guideline on thetiming of sampling. In some studies, subjects were subject to overnightfasting before blood samples were taken while in some other studies thissampling protocol was not clearly followed. Part of the confusion may berelated to the fact that most clinical guidelines for blood magnesiumtest do not require any preparation (such as fasting) for the test (see,e.g.,http://health.nytimes.com/health/guides/test/serum-magnesium-test/overview.html;http://www.med.umich.edu/1libr/aha/aha_smagnesi_crs.htm; andhttp://www.privatemdlabs.com/lp/magnesium_info.php). Thus,non-standardized sampling procedures may be a major contributing factoraccounting for the wide variations of human blood magnesium levelsreported in the literature. One aspect of the present invention providesa method for standardizing determination of physiological concentrationsof magnesium. Another aspect of the present invention is utilizing suchdeterminations to provide guidelines for magnesium supplementation toenhance beneficial effects of magnesium.

In one embodiment, the present invention provides a range ofphysiologically useful concentrations of magnesium to effect a desiredphysiological effect. In some embodiments, these concentrations are“high end” concentrations. Such “high end” concentrations include serummagnesium concentration from about 0.60 mM, 0.65 mM, 0.70 mM, 0.75 mM,0.80 mM. 0.85 mM, 0.95 mM, 1.0 mM, 1.05 mM, 1.10 mM, 1.15 mM to 1.2 mMor even higher, plasma magnesium concentration from about 0.70 mM, 0.75mM, 0.80 mM. 0.85 mM, 0.95 mM, 1.0 mM, to 1.05 mM or even higher, and/orblood ionized magnesium concentration from about 0.50 mM, 0.55 mM, 0.60mM, 0.65 mM, to about 0.70 mM. In some other embodiments, the subjectmagnesium-containing compound is effective to increase a physiologicalconcentration of magnesium by at least about 10%, 11%, 12%, 13%, 14%,15%, 20%, 25% or even higher as compared to an initial level ofmagnesium prior to administration of it to a subject. Where desired,suitable concentrations for eliciting the effects of magnesiumsupplementation as described herein can be from about 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, times the median value reported. Wheredesired, the selected physiological concentration of magnesium ismeasured under a fasting condition, e.g., without taking food for atleast about 8 hours, 10 hours, 12 hours, 15 hours, 24 hours, or evenlonger.

Additionally, magnesium compounds may be delivered to the brain of asubject via a pump or any other suitable injection device. Such devicesare known in the art and may deliver compounds directly to the brain orindirectly to the brain via the spinal cord. Administration using suchdevices, for example perispinal etanercept administration, has beendescribed previously. See, Tobinick and Gross J. Neuroinflammation 5:2).This example is given only for illustration purposes and is not intendedto be limiting on the present invention. The amount of magnesiumdelivered to the brain may be such that the magnesium concentration inthe CSF, [Mg]_(CSF), is increased by at least 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30% or more. Where desired, [Mg]_(CSF) can increase toabout 0.60, 0.65, 0.70, 0.75, 0.80. 0.85, 0.95, 1.0, 1.05, 1.10, 1.15,1.20, 1.25, 1.30, 1.35, 1.40, 1.45, or 1.5 mM. Preferably, cerebrospinalfluid concentration ([Mg]_(CSF)) is increased by at least 10%, 11%, 12%,13%, 14%, 15%, 20%, 25% or more. Where desired, [Mg]_(CSF) can beincreased to about 1.2 mM. The pump or injection device may be any knownin the art for delivering a therapeutic agent to the brain.

Magnesium is an essential mineral in the human body because of its rolesin numerous physiological functions. Yet, it is generally recognizedthat at least half of the people in the industrialized world do not getsufficient magnesium from their diets. Several diseases, such asdiabetes and Alzheimer's disease, are associated with magnesium deficit.Therefore, there is a need for magnesium supplementation. Therecommended daily allowance (RDA) for magnesium is 400 mg for adults. Byassuming that people get 40-50% of the required magnesium from diet, therecommended amount of magnesium supplement has generally been about200-250 mg per day for adults. There are numerous magnesium compoundsthat have been used as magnesium supplements. These compounds includemagnesium oxide, magnesium citrate, magnesium sulfate, magnesiumchloride, magnesium gluconate, magnesium lactate, magnesium pidolate andmagnesium diglycinate, for example. At least for nutritional purpose,the recommended amount of magnesium supplementation for most commercialmagnesium supplements is about the same (i.e., about 250 mg magnesiumper day), regardless of the bioavailability of the magnesium compoundand the individual's kidney function to retain the amount of theabsorbed magnesium. Some magnesium supplement suppliers have recommendedhigher daily magnesium intake for their products, again, withoutconsidering an individual's kidney function for magnesium retention.Similar to magnesium deficit, an excessive amount of magnesium in thebody (hypermagnesemia) may also lead to health problems, such asneuromuscular depression, hypotension, cardiac arrythmias andrespiratory paralysis. Thus, it is important to have one's bloodmagnesium level stay within the normal range. Disclosed herein is anovel method for controlling the magnesium level to a particular regionof the normal range. In some aspects of the invention, this method alsooffers particular health advantages, such as increased memorycapabilities, increased lifespan, decreased depression, and decreasedsymptoms of neurological disorders, including AD.

In addition to nutritional use, magnesium supplements have been used fortreating type 2 diabetes. In one study, diabetic patients were treatedwith nearly 1 g of magnesium daily using magnesium oxide for 1 month (deLordes Lima, et al., Diabetes Care. 21: 682-6 (1998)). The treatmentincreased the serum magnesium level of the patients by about 10% butwith only minor improvement in metabolic control. In another study,diabetic patients were treated with 720 mg/day of magnesium for threemonths. Similarly, the blood magnesium levels of the patients wereraised by about 10% on average (Eibl, et al., Diabetes Care. 21: 2031-2(1995)). However, the metabolic control of the patients, as assessed bytheir HbA1c levels, had no improvement.

Magnesium ion has been reported to be generally useful for treatment ofdementia (e.g., U.S. Pat. No. 4,985,256). Landfield and Morgan. showedthat young (9-month old) and aged (25-month old) rats fed foodcontaining 2% magnesium oxide for 8 days had shown some sign ofimprovement of cognitive function (Landfield and Morgan, Brain Research,322:167-171 (1984)). However, the gain in cognitive function wastransient and at the cost of diarrhea and weight loss to the animals. Infact, the side-effect was so severe the researchers had to use analternating feeding schedule by having the animals on the high Mg dietfor 4 days, followed by a regular diet for two days and then back to thehigh Mg diet for another 4 days.

Magnesium compounds may also be used to affect bone density. Bonedensity disorders, including but not limited to osteoporosis, may betreated by supplementation with magnesium compounds of the presentinvention. Subjects may be treated to ameliorate the effects of low bonedensity or as prophylaxis against lost bone density. Bone density may bemeasured by any means known in the art, including, but not limited to,dual energy X-ray absorptiometry (DEXA), ultrasound, quantitativecomputed tomography, single energy absorptiometry, magnetic resonanceimaging, measuring metacarpal width, and hand X-ray analysis.

As mentioned above, a magnesium-counter ion composition and/or a methoddescribed herein are useful for various purposes, such as maintaining,enhancing, and/or improving health, nutrition, and/or another conditionof a subject, and/or cognitive, learning, and/or memory function, forexample. Examples of such a condition of a subject include magnesiumdeficiency, mild cognitive impairment, Alzheimer's disease, Huntingdon'sdisease, autism, schizophrenia, cognitive decline as secondary effect ofdisease or medical treatment (HIV disease, cancer, chemotherapy),depression, dementia, attention deficit hyperactivity disorder, ALS,Parkinson's disease, diabetes, cardiovascular disease (e.g.,hypertension), glaucoma, migraine, anxiety, mood, and hypertension,merely by way of example. Magnesium supplementation may also be usefulin maintaining, enhancing, and/or improving conditions which may resultin loss of body magnesium, including, but not limited to, alcoholism,anorexia, bulemia, metabolic syndromes, and poor nutrition. Any suchcondition may be deemed or defined as a physiological, psychiatric,psychological, or medical condition or disorder, for example. Generally,the term “subject” may refer to any animal. Examples of such animalsinclude, but are not limited to, cold-blooded animals, warm-bloodedanimals, mammals, domesticated mammals, primates, humans, andindividuals or a patient to whom a composition is to be administered forexperimental, diagnostic, nutritional, and/or therapeutic purposes. Asubject or patient may be a subject or patient of normal, good, orexcellent health, mood, cognitive, and/or nutritional status, or ofcompromised health, mood, cognitive, and/or nutritional status,including of abnormal, poor, damaged, unhealthy, impaired, diseased,and/or nutritionally deficient status. The subject may be of any age,including advanced age.

Generally, the term “cognition” may refer to a process of obtaining,organizing, understanding, processing, and/or using information orknowledge. Generally, enhancing cognitive function refers to enhancingany aspect of such a process, such as learning, the performance ofmental operations, the storage, retrieval, and/or use of informationand/or thoughts, memory, and/or preventing a decline of a subjectscognitive state, for example. Various standardized tests may be used toevaluate cognition, cognitive function, and/or cognitive state and maybe used to identify a subject who might be conducive to, benefit from,and/or need, maintenance and/or enhancement of same and/or to monitor aneffect of treatment relating to same. Examples of suitable tests includethe Mini-Mental Status Exam (Folstein, 1975), components of the PROSPERneuropsychological test battery (Houx, 2002), and/or the like. Familyhistory, age, and/or other factors may also be used to identify asubject who might be conducive to, benefit from, and/or need,maintenance and/or enhancement of cognition, cognitive function, and/orcognitive state.

Generally, the term “concurrent administration” in reference to two ormore subjects of administration for administration to a subject body,such as components, agents, substances, materials, compositions, and/orthe like, refers to administration performed using dose(s) and timeintervals) such that the subjects of administration are present togetherwithin the subject body, or at a site of action in the subject body,over a time interval in less than de minimus quantities. The timeinterval may be any suitable time interval, such as an appropriateinterval of minutes, hours, days, or weeks, for example. The subjects ofadministration may be administered together, such as parts of a singlecomposition, for example, or otherwise. The subjects of administrationmay be administered substantially simultaneously (such as within lessthan or equal to about 5 minutes, about 3 minutes, or about 1 minute, ofone another, for example) or within a short time of one another (such aswithin less than or equal to about 1 hour, 30 minutes, or 10 minutes, orwithin more than about 5 minutes up to about 1 hour, of one another, forexample). The subjects of administration so administered may beconsidered to have been administered at substantially the same time. Oneof ordinary skill in the art will be able to determine appropriatedose(s) and time interval(s) for administration of subjects ofadministration to a subject body so that same will be present at morethan de minimus levels within the subject body and/or at effectiveconcentrations within the subject body. When the subjects ofadministration are concurrently administered to a subject body, any suchsubject of administration may be in an effective amount that is lessthan an effective amount that might be used were it administered alone.The term “effective amount,” which is further described herein,encompasses both this lesser effective amount and the usual effectiveamount, and indeed, any amount that is effective to elicit a particularcondition, effect, and/or response. As such, a dose of any such subjectof concurrent administration may be less than that which might be usedwere it administered alone. One or more effect(s) of any such subject(s)of administration may be additive or synergistic. Any such subject(s) ofadministration may be administered more than one time.

Generally, the term “effective amount” in reference to an active agentrefers to the amount of the active agent sufficient to elicit aparticular biological condition, effect, and/or response. The absoluteamount of a particular agent that is effective in this manner may varydepending on various factors, such as the desired biological endpoint,the agent itself, the subject or targeted part thereof, and/or the like,for example. An effective amount of an active agent may be administeredin a single dose or in multiple doses. Examples of a biologicalcondition, effect or response that may result from an effective amountof an active agent include a maintaining and/or improving of a subjectsperformance of a task involving or associated with cognitive function, amaintaining and/or improving of a subject's performance in a test thatmeasures something relating to or associated with cognitive function, amaintaining and/or improving (slowing, for example) of a rate of declinein cognitive function, and/or the like, for example. A component may bedescribed herein as having at least an effective amount, or at least anamount effective, such as that associated with a particular goal orpurpose, such as any described herein.

Generally, the term “elemental magnesium” as used in connection with amagnesium-counter ion compound described herein, may refer to a totalamount of magnesium that is present as free ion and magnesium that isbound with one or more counter ions. In general, such a term is not usedto refer to magnesium that may be associated with an agent other than amagnesium-counter ion compound that may be a component of amagnesium-counter ion composition (e.g., a pharmaceutical composition, adietary supplement composition, a foodstuff supplemented with amagnesium-counter ion compound). A small amount of magnesium may benaturally present in or otherwise associated with such an agent. Forexample, a fruit juice extract or flavoring agent may comprise an amountof magnesium from that naturally present in the fruit from which it wasderived. Generally, the term “elemental magnesium” as used in connectionwith an magnesium-counter ion compound would not encompass suchagent-associated magnesium.

As used herein, the terms “magnesium comprising component” (MCC) and“magnesium-counter ion compound” are used interchangeably, and they areuseful for purposes described herein, such as maintaining, enhancing,and/or improving health, nutrition, and/or another condition of asubject, such as magnesium deficiency, diabetes, mood, attention deficithyperactivity disorder, ALS, Parkinson's disease, anxiety, depressionand/or migraine, for example, and/or cognitive, learning, and/or memoryfunction, such as MCI and/or AD, for example.

Such a composition, such as that appropriate for administration to asubject, may comprise at least one magnesium-comprising component (MCC).The MCC may be any suitable magnesium-comprising component, such as asuitably bioavailable magnesium-comprising component. The MCC may be anysuitable biologically acceptable magnesium-comprising component. The MCCmay be any suitable organic acid magnesium salt, such as a magnesiumsalt of a non-toxic C2-C12 carboxylic acid or a magnesium salt of anon-toxic C2-C12 sulfonic acid, for example. Merely by way of example,the MCC may be a magnesium salt of an amino acid, magnesium acetate,magnesium ascorbate, magnesium citrate, magnesium gluconate, magnesiumlactate, magnesium malate, magnesium pyrrolidone carboxylate (magnesiumpidolate), magnesium taurate, and/or magnesium threonate. The at leastone MCC may be present in at least an amount effective for maintenance,enhancement, and/or treatment of health, nutrition, and/or anothercondition of a subject, and/or cognitive, learning, and/or memoryfunction, such as any of the conditions or functions described herein,for example.

In one embodiment, the composition of the invention may comprise atleast one magnesium-counter ion compound. In other embodiments, theinvention includes compositions comprising 2, 3, 4, 5, or moremagnesium-counter ion compounds. In other embodiments, the counterion(s) will be organic (e.g., threonate). In still other embodiments,the magnesium-counter ion compound has a solubility of range ofsolubility that distinguishes from Mg-gluconate/lactate/etc. In stillother embodiments, the weight % of magnesium in a magnesium-counter ioncompound is 6% or greater. In other embodiments, the weight % ofmagnesium in a magnesium-counter ion compound is 4%, 5%, 6%, 7%, 8% orgreater. In some embodiments, the organic counter ion will have 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more carbon atoms. In other embodiments, themagnesium-counter ion compound of the present invention is substantiallyfree of laxative effect.

In one embodiment, the subject magnesium-containing composition ischaracterized in that: (a) the magnesium contained therein has a weightpercentage of at least about 8%; (b) a counter ion comprises at leasttwo hydroxyl groups; (c) the composition has a solubility of at least atleast 20 mg/mL; and (d) the composition exhibit a pH value between about6 to 8.5 when dissolved in water. An example of magnesium-containingcomposition having these characteristics is one comprising magnesiumthreonate.

The magnesium-counter ion compound may be any suitably bioavailablecomposition. The magnesium-counter ion compound may be any suitablebiologically acceptable magnesium-counter ion compound. The at least onemagnesium-counter ion compound may be present in at least an amounteffective for maintenance, enhancement, and/or treatment of health,nutrition, and/or another condition of a subject, and/or cognitive,learning, and/or memory function, such as any of the conditions orfunctions described herein, for example.

A magnesium-counter ion composition may also contain a combination ofmagnesium-counter ion pairings. A magnesium-counter ion compositionappropriate for administration to a subject may also comprise an agentfor enhancing bioavailability of magnesium associated with amagnesium-counter ion compound, or a combination thereof, as furtherdescribed herein. Examples of substances which may affectbioavailability include those which affect magnesium and/or counter-ionabsorption, excretion, secretion, retention, and other physiologicallyrelevant parameters. For example, a magnesium-counter ion compositioncan comprise vitamin D3 which can reduce magnesium excretion by thekidney (Ritchie et al., Am. J. Physiol. Renal Physiol, 280:868-78(2001); Montgomery et al., J. Anim. Sci., 82:2742 (2004)), and/orvitamin E which has been suggested to promote blood magnesium enteringtissues (Barbagallo, et al., Hypertension, 34: 1002-6 (1999); Paolissoet al., Clin. Endocrinol. Metab., 85:109-15 (2000)). One of skill in theart will recognize that these two vitamins are provided only as anexample of the substances contemplated by the present invention and suchsubstances are not limited to these two vitamins.

Bioavailability of a magnesium-counter ion compound may be evaluated ormeasured in any suitable way or using any suitable criterion. Generally,bioavailability of a magnesium-counter ion compound may be evaluatedbased on magnesium absorption rate and/or magnesium loading capacity.The magnesium absorption rate refers to the fraction of a subject'smagnesium intake that is absorbed by the subject's body. In some cases,the magnesium absorption rate alone may not be sufficient to evaluatethe bioavailability of a magnesium-counter ion compound. For example,for a given magnesium-counter ion compound, the magnesium absorptionrate may stay relatively constant only when the magnesium-counter ioncomposition is administered at a relatively low dosage.

Further by way of example, for a given intake of a givenmagnesium-counter ion compound, there may be an upper limit on theamount of magnesium that can be absorbed from the magnesium-counter ioncomposition by the subject's body within a certain period, such as a24-hour period. In such a case, as the magnesium-counter ion compositiondosage increases to a certain level, the magnesium absorption rateassociated with the magnesium-counter ion composition may decline,possibly significantly. Thus, for a given magnesium-counter ioncomposition, the magnesium absorption rate may be suitable when themagnesium-counter ion composition is administered at a relatively lowdosage, but may be lower, less suitable, and/or unsuitable at arelatively high dosage.

An upper limit of the sort just described may be referred to as amagnesium loading capacity, which may be used to evaluate thebioavailability of a magnesium-counter ion compound. When amagnesium-counter ion compound that is associated with a relatively lowmagnesium loading capacity is administered to a subject at a relativelyhigh dosage in one case as compared to a relatively low dosage inanother case, the magnesium absorption rate in the one case may berelatively poorer than a magnesium absorption rate in the other case.Thus, for a magnesium-counter ion compound associated with a relativelylow magnesium loading capacity, a simple increase in dosage may beinsufficiently effective or ineffective for efficient magnesium intake,provision, and/or supplementation.

A magnesium-counter ion compound that is suitably bioavailable may beassociated with a suitable or good magnesium absorption rate and/or asuitable or good magnesium loading capacity. A magnesium-counter ioncompound of suitable bioavailability may be provided to a subject in arelatively high dosage in order to provide magnesium to a subject withsuitable speed. In some embodiments, a magnesium-counter ion compoundhaving a relatively high concentration in an aqueous medium or solventmay be orally administered to a subject for relatively rapid delivery ofmagnesium to the subject. Rapid delivery of magnesium may be importantin some cases, such as in the treatment of a subject having a severemagnesium deficit and/or another condition amenable to treatment in thismanner, for example. Oral administration may be relatively moreconvenient than intravenous injection in such cases and/or other cases.

The amount of magnesium that can be absorbed by a subject, or the rateof absorption of magnesium by a subject may vary from subject tosubject, based on any of a variety of factors. Examples of such factorsinclude metabolic rate, kidney function, overall health, and/or otherfactor(s) concerning a subject, and a property or nature of themagnesium-counter ion compound itself, such as the counter ion, anyenhancing agent, its administration vehicle or method, and/or otherfactor(s) concerning the magnesium-counter ion compound and/or itsadministration to a subject.

Determining an appropriate dosage for administration of amagnesium-counter ion compound to a subject may take into account any ofa variety of factors, such as those just mentioned, for example, anypotential or actual side-effect(s), and/or a purpose of theadministration of the magnesium-counter ion composition, such as anutritional or prophylactic purpose, a cognition maintenance orenhancement purpose, a disease or pathological condition treatmentpurpose, and/or other purpose(s) for which the magnesium-counter ioncomposition may be administered to a subject. Determining an appropriatedosage may take into account any of these factors, any other suitablefactor(s), any side-effect(s), animal study modeling, human studymodeling, clinical study modeling, drug study modeling, and anybalancing therebetween.

It is contemplated that a dosage for administration of amagnesium-counter ion compound to a subject may be from about 1.5 mg/kgof body weight/day to about 18 mg/kg of body weight/day. For example, itis contemplated that a dosage for administration of a magnesium-counterion compound to a subject may be from about 1.5 mg/kg of body weight/dayto about 9 mg/kg of body weight/day of elemental magnesium associatedwith the at least one magnesium-counter ion compound for nutritionaland/or prophylactic purpose(s); may be about 6 mg/kg of body weight/dayto about 18 mg/kg of body weight/day of elemental magnesium associatedwith the at least one counter ion for cognition maintenance and/orenhancement purpose(s); and may be about 9 mg/kg of body weight/day toabout 18 mg/kg of body weight/day of elemental magnesium associated withthe at least one counter ion for disease and/or pathological conditiontreatment purpose(s), such as the treatment of magnesium deficiency,MCI, AD, attention deficit hyperactivity disorder, ALS, Parkinson'sdisease, diabetes, migraine, depression, anxiety disorder, mooddisorder, and/or hypertension, for example. Such amounts may be suitablefor a human subject, for example.

As mentioned above, such a dosage may be determined, modified and/orrefined based on any suitable factor(s), such as results of clinicaltrials concerning subjects, for example human subjects. In someembodiments, a suitable dosage may be determined, modified and/orrefined based on a determination of a suitable dosage for a suitableanimal model, based on experimental studies or tests, for example, andconversion of such a suitable animal dosage to a suitable human dosage,based on suitable conversion factor(s), such as any suitable establishedconversion factor(s), for example. Further by way of example, it iscontemplated that any such suitable human dosage may be furtherdetermined, modified and/or refined based on clinical trials involvinghuman subjects, for example.

As mentioned above, a magnesium-counter ion composition appropriate foradministration to a subject may also comprise at least one agent(“enhancing agent”) for enhancing bioavailability of magnesiumassociated with a counter ion of the composition or more than onecounter ion of the composition. The enhancing agent may be any suitableagent, such as a biologically acceptable agent. Merely by way ofexample, a mass ratio of an amount of elemental magnesium associatedwith the at least one counter ion and an amount of the at least oneenhancing agent may be from about 1 to about 5 (˜1:˜5) to about 1 toabout 3000 (˜1:˜3000); or from about 1 to about 10 (˜1:˜10) to about 1to about 1000 (˜1:˜1000); or from about 1 to about 200 (˜1:˜200) toabout 1 to about 3000 (˜1:˜3000). Herein, such a mass ratio refers to aratio of a total mass of a single magnesium-counter ion compound, ifonly one is present in the composition, or of multiple magnesium-counterion compounds, if more than one are present in the composition, to atotal mass of a single enhancing agent, if only one is present in thecomposition, or of multiple enhancing agents, if more than one arepresent in the composition.

Merely by way of example, a magnesium-comprising composition appropriatefor administration to a subject may comprise at least one MCC and atleast one component of non-acidified milk sufficient to enhancebioavailability of magnesium associated with at least one MCC. Acomponent or several components of non-acidified mammalian milk otherthan water, such as lactose, a fatty acid or milk fat thereof, and/oranother organic component thereof, for example, may enhance thebioavailability of magnesium associated with an MCC or more than oneMCC. The mammalian milk source of such a component or such componentsmay be that having its original amount of milk fat, such as a naturallyoccurring amount of milk fat, for example, or an amount of milk fat thatis less than its original amount of milk fat, such as a manipulated orartificially reduced amount of milk fat. Accordingly, a component, suchas a fatty acid component, for example, may be more or less fatty and/orhave a greater or lesser chain length, for example. The mammalian milksource of such a component or such components may be non-acidified, asacidification, such as that associated with fermentation, for example,may alter the component or the components such that magnesiumbioavailability is not enhanced or not sufficiently enhanced by thepresence of the component or the components in the composition. Merelyby way of example, while lactose may be a suitable enhancement agent,lactic acid, a product of lactose acidification, may not. Merely by wayof example, a suitable non-acidified mammalian milk source may have a pHof from about 5.7 to about 7.2.

Merely by way of example, a magnesium-comprising composition appropriatefor administration to a subject may comprise at least one MCC andlactose, the latter of which may act as an enhancing agent. In such acase, the mass ratio of an amount of elemental magnesium associated withthe at least one MCC to an amount of lactose may be from about 1 toabout 10 (˜1:˜10) to about 1 to about 1000 (˜1:˜1000). Further, merelyby way of example, a magnesium-comprising composition appropriate foradministration to a subject may comprise at least one MCC and thecomplete organic components, excluding water, of non-acidified milk, thelatter of which may comprise an enhancing agent or enhancing agents. Insuch as case, the mass ratio of elemental magnesium associated with theat least one MCC to the enhancing agent(s) may be from about 1 to about200 (˜1:˜200) to about 1 to about 3000 (˜1:˜3000).

As described above, a magnesium-comprising composition appropriate foradministration to a subject may comprise at least one MCC, such asmagnesium gluconate, magnesium lactate, and/or magnesium citrate, forexample. Each of magnesium gluconate, magnesium lactate, and magnesiumcitrate is commercially available and relatively palatable. An MCC, orcomposition comprising same, that is tolerably or relatively palatablemay be used in a food, a beverage, and/or another type of consumablevehicle that may be associated with a diet of a subject, such as a humansubject, for example. As such, the subject may be able to provide and/orsupplement a normal magnesium intake via a diet comprising at least onesuch magnesium-comprising consumable vehicle, rather than via arelatively non-dietary means, such as at least one magnesium-containingpill, capsule, and/or tablet, for example. Naturally, a subject mayemploy one or more than one means of magnesium intake, provision, and/orsupplementation.

As also described above, a magnesium-comprising composition appropriatefor administration to a subject may comprise more than one MCC, or acombination of MCCs. Merely by way of example, such amagnesium-comprising composition may comprise at least two MCCs, such asat least two MCCs of any of the MCCs described herein. Further, merelyby way of example, a magnesium-comprising composition may comprise atleast two MCCs selected from magnesium gluconate, magnesium lactate,magnesium citrate, and magnesium malate, for example, or selected frommagnesium gluconate, magnesium lactate, and magnesium citrate, forexample, such as all three of magnesium gluconate, magnesium lactate,and magnesium citrate, for example. Still further, merely by way ofexample, a magnesium-comprising composition may comprise magnesiumlactate in an amount from about 5 to about 50%, such as about 25%, forexample; magnesium citrate in an amount of from about 5 to about 50%,such as about 25%, for example; and/or magnesium gluconate in an amountfrom about 10 to about 70%, such as about 50%, for example, where allpercentages are weight percentages relative to the total weight of anyof these three MCCs present. Any such composition may also comprise anysuitable enhancing agent, such as any described herein, for example.

Magnesium lactate is associated with a relatively good magnesium contentof about 12 percent by weight. Magnesium citrate is associated with arelatively good magnesium content of about 18.46 percent by weight.While magnesium gluconate is associated with a comparatively lowermagnesium content of about 5.86 percent by weight and comparativelylower palatability, particularly at high concentration, it is alsoassociated with a solubility in water or an aqueous medium that iscomparatively better than that associated with either magnesium lactateor magnesium citrate. As described above, a magnesium-comprisingcomposition may comprise at least two MCCs selected from magnesiumgluconate, magnesium lactate, and magnesium citrate, such as all threeof these MCCs, for example.

A magnesium-counter ion composition comprising more than onemagnesium-counter ion compound may be suitable, beneficial or desirablerelative to a magnesium-counter ion composition comprising a singlemagnesium-counter ion compound. A combination of more than onemagnesium-counter ion compound may be suitable, beneficial or desirablein terms of any number of features or factors, such as magnesiumcontent, solubility, palatability, magnesium bioavailability, biologicalacceptability, and/or the like, for example. A combination of more thanone magnesium-counter ion compound may be suitable, beneficial ordesirable in terms of palatability. A combination of more than onemagnesium-counter ion compound may be suitable, beneficial or desirablein terms of maintaining and/or enhancing an attribute or attributes of amagnesium-counter ion compound or several magnesium-counter ioncompounds.

In terms of solubility, a magnesium-counter ion compound, or more thanone magnesium-counter ion compound, may have solubility in water of atleast about 20 mM, such as at least about 50 mM or at least about 80 mM,merely by way of example. In terms of magnesium content, anmagnesium-counter ion compound or more than one magnesium-counter ioncompound may have a magnesium content of at least about 8 weightpercent. In terms of bioavailability, a magnesium-counter ion compoundor more than one magnesium-counter ion compound may be associated with abioavailability that is at least comparable to that associated withmagnesium chloride, if not greater.

A magnesium-comprising composition comprising at least one MCC and anenhancing agent may be associated with suitable magnesiumbioavailability. Such a composition may be associated with a suitablemagnesium absorption rate. By way of example, when rats were feddifferent compositions comprising magnesium gluconate, at aconcentration of 12 mM, in different media, namely, skim milk, watercomprising 5 weight percent by lactose, milk prepared from powdered milkand water, milk cream and water, and a control medium of water,respectively, each of the four compositions outperformed the controlcomposition in terms of magnesium absorption rate. Further, asgraphically depicted in FIG. 2 and described in Example 3, each of thecompositions comprising a medium other than the control mediumoutperformed the composition comprising the control medium, water, interms of the percentage of magnesium absorption rate enhancement.Further by way of example, when rats were fed a composition comprising acombination of magnesium gluconate, magnesium lactate, and magnesiumcitrate, and skim milk, the composition was associated with a suitablemagnesium absorption rate, one that was higher than that associated witha control composition comprising the same combination of magnesiumgluconate, magnesium lactate, and magnesium citrate, but water in placeof skim milk, as graphically depicted in FIG. 3 and described in Example4. Further by way of example, when rats were fed compositions comprisingmagnesium gluconate, at various relatively low magnesium dosages, andeither skim milk or water comprising 5 weight percent lactose, thecompositions were associated with suitable magnesium absorption rates,as graphically depicted in FIG. 4 and described in Example 5.

A magnesium-counter ion composition comprising at least one counter ionand an enhancing agent may be associated with a suitable magnesiumloading capacity, such as a relatively high loading capacity, forexample. Such a composition may be associated with a relatively highmagnesium absorption rate, for example, throughout a relatively widedosage range. When such a composition is administered to a subject in arelatively high dosage, the subject may be able to absorb a suitableamount of magnesium, such as a nutritional, therapeutic, and/orprophylactic amount, or may be able to do so in a relatively shortperiod. By comparison, when a composition associated with a lowmagnesium loading capacity is administered to a subject in a relativelyhigh dose, the subject may not be able to absorb a suitable amount ofmagnesium, such as a nutritional, therapeutic, and/or prophylacticamount, or may not be able to do so in a relatively short period. Thatis, in the latter case, simply administering a large dosage of acomposition associated with a low magnesium loading capacity to asubject may not be sufficient or effective for a particular purpose. Byway of example, when rats were fed compositions comprising magnesiumgluconate, at a relatively low magnesium dosage and at a relatively highmagnesium dosage, and either skim milk or water comprising 5 weightpercent lactose, the lower dosage compositions were associated withsuitable magnesium absorption rates and the higher dosage compositionswere associated with suitable magnesium absorption rates that weresuitably close to those associated with the lower dosage compositions,as graphically depicted in FIG. 4 and described in Example 5. Thesemagnesium gluconate-comprising compositions were thus associated withsuitable magnesium loading capacities. A composition comprisingmagnesium gluconate and milk, lactose, or another enhancing agent, whenadministered at high dosage, may thus be suitable for rapid and/orefficient magnesium intake, provision, and/or supplementation. By way ofcomparison, when rats were fed compositions comprising magnesiumchloride, at a relatively low magnesium dosage and at a relatively highmagnesium dosage, and water, the lower dosage compositions wereassociated with suitable, but lower, magnesium absorption rates and thehigher dosage compositions were associated with magnesium absorptionrates that were less desirable, as graphically depicted in FIG. 4 anddescribed in Example 5. Thus, while magnesium chloride has previouslybeen associated with very good bioavailability, that level ofbioavailability may be associated with a relatively low dosage, and notwith a relatively high dosage. A composition comprising magnesiumchloride and water, when administered at high dosage, may thus be lessdesirable or suitable, and perhaps unsuitable, for rapid and/orefficient magnesium intake, provision, and/or supplementation.

A magnesium-counter ion compound appropriate for administration to asubject may comprise magnesium threonate, in which each magnesium cationis associated with two threonate anions, as illustrated in the formulaprovided below.

Such a composition may be prophylactically and/or therapeuticallysuitable or beneficial. Threonate is a natural metabolic product ofvitamin C or ascorbic acid that may be associated with non-toxicity inanimals (Thomas et al, Food Chem. 17, 79-83 (1985)) and biologicalbenefit, such as the promotion of vitamin C uptake, in animals(Verlangieri et al., Life Sci. 48, 2275-2281 (1991)).

Magnesium threonate may be associated with suitable magnesiumbioavailability in relation to a subject. As such, a magnesium-counterion composition appropriate for administration to a subject may comprisemagnesium threonate, and optionally, an enhancing agent. By way ofexample, when rats were fed a relatively dilute composition comprisingmagnesium threonate and water, at a relatively low dosage, thecomposition was associated with a suitable magnesium absorption rate, asgraphically depicted in FIG. 5 and described in Example 6. As shown, themagnesium absorption rate of this composition was similar to thatassociated with a similarly tested composition comprising magnesiumchloride and water, at a relatively low dosage, as graphically depictedin FIG. 5 and described in Example 6. When rats were fed a compositioncomprising magnesium threonate and water, at a higher dosage, thecomposition was still associated with a suitable absorption rate, asgraphically depicted in FIG. 5 and described in Example 6. As shown, themagnesium absorption rate of this composition was significantly higherthan that associated with a similarly tested composition comprisingmagnesium chloride and water, at a higher dosage, as graphicallydepicted in FIG. 5 and described in Example 6. A composition comprisingmagnesium threonate may thus be associated with a suitable magnesiumloading capacity and may be suitable for rapid and/or efficientmagnesium intake, provision, and/or supplementation.

Magnesium threonate may be more suitable or desirable for oraladministration to a subject than some other magnesium-counter ioncompounds, such as various inorganic magnesium compounds and variousmagnesium chelates. The oral administration of various inorganicmagnesium compounds, such as magnesium chloride and magnesium sulfate,for example, at high dosages, may contribute or lead to diarrhea, alaxative effect, and/or the like. In view of the laxative effect ofmagnesium sulfate on the digestive system, magnesium sulfate may beadministered by intravenous injection for non-laxative purposes in orderto avoid the digestive system altogether. Further, oral administrationof various magnesium chelates, such as magnesium diglycinate, may becomplicated by alkalinity and/or palatability concerns. A magnesiumchelate may comprise one magnesium ion associated with one amino acidmolecule or two amino acid molecules and may be associated withrelatively high bioavailability. A magnesium chelate may be highlyalkaline at a pH of 10 or more when dissolved in water. A magnesiumchelate may be associated with a smell or a taste like that associatedwith rotten fish, perhaps reflecting that the amine groups thereof arerelatively free as opposed to stably bonded in relation to themagnesium. In view of alkalinity, sensory and/or palatability concernsthat may be associated with a magnesium chelate, such compounds may benot be the most suitable for magnesium intake, provision, and/orsupplementation via a consumable vehicle or oral administration.

Magnesium threonate does not present the challenges that may beassociated with various inorganic magnesium compounds and variousmagnesium chelates. A composition comprising magnesium threonate wasshown to have a more suitable magnesium loading capacity than acomposition comprising magnesium chloride, as described in relation toFIG. 5 and Example 6. Briefly, ten adult male rats were fed a magnesiumthreonate solution having a magnesium threonate concentration of 48 mMover a three-month period, for an average magnesium dosage of 40 mg/kgof body weight/day, they did not show signs of diarrhea. Still further,when rats were exposed to a diet including a magnesium-counter ioncomposition of magnesium threonate in water, their serum magnesiumconcentration was greater than that associated with rats that wereexposed to a diet including either of two other magnesium-counter ioncompositions, or a diet including de-ionized water, as graphicallydepicted in FIG. 6 and described in Example 7. A magnesium-counter ioncompound sufficient to produce a relative high magnesium concentrationin blood (e.g., magnesium threonate) may be useful in any of a varietyof applications, such as a therapeutic application, for example.

Magnesium threonate may be suitable for relatively rapid magnesiumintake, provision, and/or supplementation, as may be suitable orbeneficial for any of a variety of applications, such as a nutritionalor prophylactic application, and/or a therapeutic application. Magnesiumthreonate may be a suitable or beneficial vehicle for magnesium intake,provision, and/or supplementation application(s), such as any that maybe accomplished via a dietary vehicle or a consumable vehicle, such as amagnesium-fortified food and/or a magnesium-fortified beverage, forexample.

A magnesium-counter ion compound appropriate for administration to asubject may be useful in nutritional applications and/or therapeuticapplications. A nutritional application may refer to an applicationsuitable for warding off and/or preventing pathological condition and/ordisease associated with magnesium deficit and/or subject to treatmentwith magnesium, such as AD, MCI, and/or diabetes. A nutritionalapplication may refer to an application suitable for maintaining and/orenhancing physiological function, such as physiological function at astate considered normal. A level of cognitive function, such as learningor memory function, for example, of a healthy human may be maintainedand/or enhanced by administering a suitable magnesium-counter ioncomposition. A therapeutic application includes, but is not limited to,treating pathological condition and/or disease associated with magnesiumdeficit and/or subject to treatment with magnesium, such as AD, MCI,ALS, Parkinson's disease, diabetes, and/or hypertension.

A magnesium-counter ion compound, such as magnesium threonate, and/or acomposition comprising one or more magnesium-counter ion compounds, maybe sufficient to at least maintain and/or to enhance cognitive function.In such a composition, an amount of magnesium, or an effective amount ofsame, associated with at least one magnesium-counter ion compound may besufficient for any suitable function described herein. For example, aconcentration of elemental magnesium associated with at least onecounter ion of such a composition in a liquid form (e.g., an aqueoussolution) may be from about 5 mg/L to about 12 g/L, such as from about50 mg/L to about 12 g/L, for example.

A magnesium-counter ion compound, such as magnesium threonate and/or acomposition comprising one or more counter ions, may be sufficient fortreating MCI, AD, and/or any other suitable malady or disease. In such acomposition, an amount of magnesium, or an effective amount of same,associated with at least one magnesium-counter ion component may besufficient for any suitable function described herein. For example, aconcentration of elemental magnesium associated with at least onecounter ion of such a composition in a liquid form (e.g., an aqueoussolution) may be from about 5 mg/L to about 12 g/L, such as from about50 mg/L to about 12 g/L, for example.

A subject afflicted with AD may have trouble carrying out a task, suchas speaking, understanding, writing, reading, grooming, drinking, oreating, for example, either with or without assistance. Before now, ADhas been considered an incurable disease that typically becomes worseover time. Various drugs that have been used to treat AD have beendesigned to slow its progression. Some of these drugs have beenassociated with various side-effects, some of which may be significantor serious. A subject afflicted with MCI may experience forgetfulnessthat can affect daily life. Before now, no treatment has been availablespecifically for MCI, which may progress into AD. Various drugs thathave been used to treat AD may not be suitable for treating the milderdisease, MCI, in view of associated side-effects. A magnesium-counterion compound, such as magnesium threonate, for example, and/orcomposition comprising one or more magnesium-counter ion compounds, maybe sufficient for any suitable purpose described herein, such astreating AD and/or MCI and/or ameliorating a symptom associatedtherewith, for example, while not giving rise to an undesirableside-effect of significance.

In some embodiments, the magnesium-counter ion compounds of the presentinvention may be administered to a subject to address cognitivefunction, whether nutritionally or prophylactically or therapeutically,in any suitable manner. As graphically depicted in FIG. 7 and describedin Example 8, AD-afflicted mice fed a magnesium-fortified diet for overa month were shown to have improved short-term spatial memory andlearning capacity, relative to AD-afflicted mice fed a normal diet.

A magnesium-counter ion compound described herein may be administered toa subject, whether or not afflicted with cognitive decline, deficiency,and/or impairment, to address cognitive function, whether nutritionallyor prophylactically or therapeutically, in any suitable manner. Forexample, such compounds may be administered to a relatively young and/orhealthy subject. A magnesium-counter ion compound described herein maybe administered to a subject to achieve its purpose, such as addressingof cognitive function in any suitable manner, in a relatively shortperiod. As graphically depicted in FIG. 8 and described in Example 9,young rats, none of which had been associated with cognitive decline,deficiency, and/or impairment, fed a magnesium-fortified diet over timewere shown to have markedly improved over time in terms of enhancementof spatial working memory and learning. In contrast, such rats fed anormal diet over time were generally shown not to have improved in thismanner over time. Further, the rats that showed marked improvement didso over a period of less than two weeks.

It is contemplated that a magnesium-counter ion compound describedherein may be administered to a human subject to suitable or beneficialeffect, such as nutritional, prophylactic, and/or therapeutic effect,for example, as may be useful to address cognitive function, forexample, in any suitable manner. In some embodiments, amagnesium-counter ion compound of the present invention may beadministered to a human subject susceptible to, or afflicted by, MCIand/or AD to suitable or beneficial effect. In other embodiments amagnesium-counter ion compound, or a composition containing such acompound, may be administered to a human subject for a variety of usefulpurposes, such as the maintenance, enhancement, and/or improvement ofcognitive function, learning, memory, mood, anxiety, depression,migraine, and/or other conditions. As the magnesium-counter ioncomposition comprises an endogenous mineral, magnesium, and possiblyother natural ingredients, such as an enhancing agent described herein,for example, in most embodiments administration of the magnesium-counterion compounds of the present invention may be safe over a relativelylong term. In still other embodiments, administration of such amagnesium-counter ion compound or composition occurs over a long-termperiod. For example, a subject may be administered the compound and/orcompositions of the present invention for weeks, months, years, and/orfor life. Such long-term administration may be used for preventing ortreating a condition, such as MCI, or may be useful for preventingprogression of a condition (e.g., preventing the progression of acondition, such as MCI, into another condition, such as AD). Theseexamples are not limiting examples, as long-term administration of themagnesium-counter ion compounds of the present invention may be used formultiple purposes as described herein and as recognized by one of skillin the art.

A magnesium-counter ion composition described herein may comprise one ormore other suitable component(s), such as a suitable pharmaceuticalcomposition or drug associated with the treatment of MCI, AD, diabetes,ADHD, ALS, Parkinson's disease, ALS, and/or hypertension, for example.Magnesium, particularly in the form of a magnesium-counter ion compoundof the present invention (e.g., magnesium threonate) may be effective inthe treatment of hypertension. A subject afflicted with MCI, AD, and/ordiabetes may have a magnesium deficiency, which may be addressed by apharmaceutical composition drug used to treat the affliction. It iscontemplated that magnesium and such a pharmaceutical composition ordrug in a magnesium-counter ion composition described herein may worksynergistically in a suitable manner, such as a biologically beneficialand/or a therapeutically effective manner. Non-limiting examples of apharmaceutical composition or drug associated with the treatment of ADinclude acetylcholine esterase inhibitors, (e.g., donepezil,rivastagmine, or galantamine) and NMDA channel blockers, such asmemantine. One of skill in the art will recognize that thesepharmaceuticals are given merely by way of example and do not delineatethe scope of pharmaceuticals which may be used in combination with themagnesium-counter ion compounds of the present invention.

A magnesium-counter ion compound appropriate for administration to asubject may be administered in any suitable manner. Such administrationmay be oral and/or any other suitable administration, such astransdermal, intramuscular, vaginal, rectal, subdermal. Components of amagnesium-counter ion composition, such as at least onemagnesium-counter ion compound and at least one agent for enhancingbioavailability of magnesium may be administered to a subjectconcurrently, such as in any manner of concurrent administrationdescribed herein and/or in U.S. Patent Application Publication No. US2006/0089335 A1.

A magnesium-counter ion compound appropriate for administration to asubject may be provided in any suitable form, such as a liquid form, agel form, a semi-liquid (for example, a liquid, such as a viscousliquid, containing some solid) form, a semi-solid (a solid containingsome liquid) form, and/or a solid form, for example. Merely by way ofexample, a tablet form, a capsule form, a food form a chewable form, anon-chewable form, a slow- or sustained-release form, a non-slow- ornon-sustained-release from, and/or the like, may be employed.Gradual-release tablets are known in the art. Examples of such tabletsare set forth in U.S. Pat. No. 3,456,049. Such a composition maycomprise an additional agent or agents, whether active or passive.Examples of such an agent include a sweetening agent, a flavoring agent,a coloring agent, a filling agent, a binding agent, a lubricating agent,an excipient, a preservative, a manufacturing agent, and/or the like,merely by way of example, in any suitable form. A slow- orsustained-release form may delay disintegration and/or absorption of thecomposition and/or one or more component(s) thereof over a period, suchas a relatively long period, for example. A food form may take the formof a food bar, a cereal product, a bakery product, a dairy product,and/or the like, for example. A bakery product form may take the form ofa bread-type product, such as a bagel or bread itself, for example, adonut, a muffin, and/or the like, merely by way of example. A componentof a magnesium-counter ion composition may be provided in a form that isother than that of another component of the magnesium-counter ioncomposition. For example, at least one magnesium-counter ion compoundmay be provided in a solid form, such as solid food or cereal that istaken with an enhancing agent in a liquid form, such as a liquid dietarysubstance. Such administration of magnesium-counter ion compositions inmultiple forms, may occur simultaneously (e.g., ingesting a magnesiumthreonate tablet with magnesium threonate-fortified milk), or atdifferent times.

In some embodiments, a magnesium-counter ion composition in the form ofa pill, tablet, capsule, or like device, may comprise from about 30 mgto about 200 mg of elemental magnesium. In other embodiments, amagnesium-counter ion composition may contain from about 50 mg to about100 mg of elemental magnesium associated with the at least onemagnesium-counter ion compound. In still other embodiments, amagnesium-counter ion composition in the form of a food serving, or likedietary serving, may comprise from about 20 mg to about 1 g or even 1.5g of elemental magnesium. In still other embodiments, amagnesium-counter ion composition in the form of a food serving, or likedietary serving, may comprise from about 50 mg to about 800 mg ofelemental magnesium.

A magnesium-counter ion composition appropriate for administration to asubject may be provided in a liquid form, such as one suitable for oraladministration, parenteral administration and/or other appropriateroutes. Such a composition may comprise any suitable additional agent oragents, whether active or passive. Examples of such agents includewater, a sweetening agent, a flavoring agent, a coloring agent, atexturing agent, a stabilizing agent, a preservative, a manufacturingagent, and/or the like, in any suitable form. A component that maynegatively affect magnesium bioavailability, such as a phosphate or apolyphosphate, for example, may be avoided. A magnesium-counter ioncomposition in a liquid form may comprise from about 5 mg/L to about 12g/L, such as from about 50 mg/L to about 12 g/L, for example, ofelemental magnesium associated with the magnesium-counter ion of thecomposition. An amount of from about 50 mg/L to about 3 g/L, such asfrom about 100 mg/L to about 1.5 g/L, for example, of elementalmagnesium associated with the magnesium-counter ion may be suitable forprophylactic application and/or nutritional application. An amount offrom about 300 mg/L to about 12 g/L, such as from about 500 mg/L toabout 3.5 g/L, for example, of elemental magnesium associated with themagnesium-counter ion may be suitable for therapeutic application.

A magnesium-counter ion composition in a liquid form may be used in anysuitable manner. In some embodiments, the magnesium-counter ioncomposition may be used as a beverage, such as a milk-based beverage, asports drink, a fruit juice drink, an alcoholic beverage, and/or thelike. In other embodiments, the magnesium-counter ion composition inliquid form contains multiple magnesium-counter ion compounds. In suchembodiments, the weight percentage of each magnesium-counter ioncompound may vary in relation to the other. In still other embodiments,the magnesium-counter ion composition in a liquid form may take the formof a magnesium-fortified product comprising water, magnesium threonate,and optionally, at least one agent sufficient to confer a suitableproperty to the product. In still another embodiment, amagnesium-counter ion composition in a liquid form may be formulatedfrom a dry mix, such as a dry beverage mix or a magnesium-fortified,milk-comprising powder. A dry mix may be suitable in terms oftransportation, storage, and/or shelf life. The composition may beformulated from the dry mix in any suitable manner, such as by adding asuitable liquid (e.g., water, milk, fruit juice, alcohol, etc.).

Examples concerning magnesium-counter ion compound(s) andmagnesium-counter ion composition(s), and the preparation, testingand/or use of same, are provided below.

Use as Dietary Supplement

One embodiment of the present invention is a magnesium dietarysupplement. In some embodiments, the magnesium supplement contains oneor more magnesium-counter ion compounds of the present invention and mayoptionally contain other ingredients generally recognized as safe forfood additive use, including, but not limited to, preservatives (e.g.,butylated hydroxytoluene, butylated hydroxyanisole), food gradeemulsifiers (e.g., lecithin, propylene glycol esters), andpharmaceutically acceptable carriers and excipients (e.g., binders,fillers, lubricants, dissolution aids).

In one embodiment, the magnesium-counter ion supplement composition ofthe present invention is made by combining magnesium threonate or othermagnesium compounds of the invention, as well as any optionalcomponents, in the desired relative amounts and mixing the componentsaccording to known methods to produce a substantially homogeneousmixture.

In another embodiment, the magnesium-counter ion composition may alsocontain other nutritional active materials including, withoutlimitation, calcium-containing materials such as calcium carbonate,stannol esters, hydroxycitric acid, vitamins, minerals, herbals, spicesand mixtures thereof. Examples of vitamins that are available asadditional ingredients include, but are not limited to, vitamin A(retinol), vitamin D (cholecalciferol), vitamin E group(alpha-tocopherol and other tocopherols), vitamin K group(phylloquinones and menaquinones), thiamine (vitamin B₁), riboflavin(vitamin B₂), niacin, vitamin B₆ group, folic acid, vitamin B₁₂(cobalamins), biotin, vitamin C (ascorbic acid), and mixtures thereof.The amount of vitamin or vitamins present in the final product isdependent on the particular vitamin. Examples of minerals that areavailable as additional ingredients include, but are not limited to,calcium, magnesium, phosphorus, iron, zinc, iodine, selenium, potassium,copper, manganese, molybdenum and mixtures thereof. As is the case withvitamins, the amount of mineral or minerals present in the final productis dependent on the particular mineral. It will be clear to one of skillin the art that the present list of additional neutriceutical componentsare provided by way of example only, and are not intended to belimiting.

Magnesium threonate is a highly bioavailable form of a magnesiumcounter-ion composition. However, the in vivo accessibility of thismagnesium threonate may be provided in multiple ways. In someembodiments, a subject ingests magnesium threonate. In otherembodiments, magnesium may be taken with other supplements which resultin an in vivo reconstitution of magnesium-counter ion composition.Without being bound by theory, the threonate may function to promotecellular uptake of magnesium in any form and may also enhance deliveryto the brain and central nervous system. Thus, in some embodiments,magnesium may be given uncomplexed with threonate and threonate isprovided to the same subject to enhance absorption. For example,magnesium gluconate and potassium threonate may be taken essentiallyconcurrently to result in an in vivo reconstitution of magnesiumthreonate and/or enhance magnesium uptake and/or delivery of magnesiumto the brain. In another example, certain counter ions may be metabolicproducts of other substances. For example, vitamin C is metabolized intothe threonate ion in humans; therefore, ingestion of magnesium in a formwhich can be taken up by the body and vitamin C may result in thereconstitution of magnesium threonate in the body. Another example of asubstance which is metabolized to threonate in humans is ascorbate.Thus, in some embodiments of the present invention, magnesium ascorbatemay be provided to a subject and this substance would be metabolized tomagnesium and threonate in vivo. One of skill in the art will recognizethat these examples are provided by way of illustration only and thatother combinations of magnesium compounds and secondary compounds mayresult in the reconstitution of a magnesium-counter-ion composition invivo.

In yet another embodiment, the present dietary supplement or foodcompositions are formulated to have suitable and desirable taste,texture, and viscosity for consumption. Any suitable food carrier can beused in the present food compositions. Food carriers of the presentinvention include practically any food product. Examples of such foodcarriers include, but are not limited to food bars (granola bars,protein bars, candy bars, etc.), cereal products (oatmeal, breakfastcereals, granola, etc.), bakery products (bread, donuts, crackers,bagels, pastries, cakes, etc.), beverages (milk-based beverage, sportsdrinks, fruit juices, alcoholic beverages, bottled waters), pastas,grains (rice, corn, oats, rye, wheat, flour, etc.), egg products, snacks(candy, chips, gum, chocolate, etc.), meats, fruits, and vegetables.

In an embodiment, food carriers employed herein can mask the undesirabletaste (e.g., bitterness), if present in one or more of the subjectmagnesium-counter ion compounds. Where desired, the food compositionpresented herein exhibit more desirable textures and aromas than that ofthe magnesium-counter ion compounds.

For example, liquid food carriers may be used according to the inventionto obtain the present food compositions in the form of beverages, suchas supplemented juices, coffees, teas, and the like. In otherembodiments, solid food carriers may be used according to the inventionto obtain the present food compositions in the form of mealreplacements, such as supplemented snack bars, pasta, breads, and thelike. In yet other embodiments, semi-solid food carriers may be usedaccording to the invention to obtain the present food compositions inthe form of gums, chewy candies or snacks, and the like

In another embodiment, the supplement composition of the presentinvention may be administered in any oral dosage form, including liquiddosage forms (e.g., a suspension or slurry), and oral solid dosage forms(e.g., a tablet or bulk powder). As used herein the term “tablet” refersgenerally to tablets, caplets, capsules, including soft gelatincapsules, and lozenges.

Tablets are made by methods known in the art and may further comprisesuitable binders, lubricants, diluents, disintegrating agents,colorants, flavoring agents, flow-inducing agents, melting agents whichare known in the art. The oral solid dosage form may, optionally, have afilm coating to protect the components of the magnesium-counter ionsupplement composition from one or more of moisture, oxygen and light orto mask any undesirable taste or appearance. Suitable coating agentsinclude, for example, cellulose, hydroxypropylmethyl cellulose. Wheredesired, tablets can be formulated in sustained release format. Methodsof making sustained release tablets are known in the art, e.g., seeUS2006051416 and US20070065512, both of which are incorporated herein byreference.

In still other embodiments, magnesium-counter ion compounds of thepresent invention are added to foodstuffs. Such foodstuffs may benaturally high or low in magnesium. Examples of foodstuffs which arehigh in magnesium include, but are not limited to soft drinks (e.g.,coke, gaterade, coffee), milk, bran flakes, oatmeal, shredded wheat,whole wheat bread, fruit and/or vegetable juices, and potatoes. Otherfoodstuffs are readily apparent and multiple examples have beendescribed. See, e.g., U.S. Pat. Nos. 6,790,462, 6,261,589, and U.S.patent application Ser. Nos. 10/725,609 and 11/602,126.

Use as Pharmaceutical

One embodiment of the present invention is a pharmaceutical composition,typically for administration to a person in need of therapeutic levelsof magnesium. Various delivery systems are known and can be used toadminister the magnesium compositions of the invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, etc. Methodsof delivery include but are not limited to intra-arterial,intramuscular, intravenous, intranasal, and oral routes. In a specificembodiment, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,transdermal patches, local infusion during surgery, by injection, bymeans of a catheter (with or without an attached pump), or bathing in amagnesium solution. In some embodiments, the agents are delivered to asubject's nerve systems, preferably the central nervous system.

In some embodiments, administration of the magnesium-counter ioncompositions can be effected in one dose, continuously or intermittentlythroughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the composition used for therapy,the purpose of the therapy, the target cell or tissue being treated, andthe subject being treated. Single or multiple administrations can becarried out with the dose level and pattern being selected by thetreating physician.

For oral administration, the inventive compositions may optionally beformulated by mixing the magnesium-containing compositions withphysiologically or pharmaceutically acceptable carriers that are wellknown in the art. Such oral dosage forms may be formulated as tablets,pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by an individual or a patient to be treated.

In one embodiment, the magnesium-containing composition is contained incapsules. Capsules suitable for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astale or magnesium stearate and, optionally, stabilizers. Optionally, theinventive composition for oral use can be obtained by mixing themagnesium-containing composition with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For buccal administration, theinventive compositions may take the form of tablets or lozengesformulated in a conventional manner. For administration by inhalation,the compositions of the present invention may be delivered in the formof an aerosol spray presentation from pressurized packs or a nebulizer,with the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The preparation of pharmaceutical compositions of this invention isconducted in accordance with generally accepted procedures for thepreparation of pharmaceutical preparations. See, for example,Remington's Pharmaceutical Sciences 18th Edition (1990), E. W. Martined., Mack Publishing Co., PA. Depending on the intended use and mode ofadministration, it may be desirable to process the magnesium-counter ioncompound further in the preparation of pharmaceutical compositions.Appropriate processing may include mixing with appropriate non-toxic andnon-interfering components, sterilizing, dividing into dose units, andenclosing in a delivery device.

Pharmaceutical compositions for oral, intranasal, or topicaladministration can be supplied in solid, semi-solid or liquid forms,including tablets, capsules, powders, liquids, and suspensions.Compositions for injection can be supplied as liquid solutions orsuspensions, as emulsions, or as solid forms suitable for dissolution orsuspension in liquid prior to injection. For administration via therespiratory tract, a preferred composition is one that provides a solid,powder, or aerosol when used with an appropriate aerosolizer device.

Liquid pharmaceutically acceptable compositions can, for example, beprepared by dissolving or dispersing a polypeptide embodied herein in aliquid excipient, such as water, saline, aqueous dextrose, glycerol, orethanol. The composition can also contain other medicinal agents,pharmaceutical agents, adjuvants, carriers, and auxiliary substancessuch as wetting or emulsifying agents, and pH buffering agents.

In some embodiments, magnesium supplementation is provided to achieveoptimal body magnesium status by supplementing a person's diet with amagnesium composition of the present invention. As described herein,there is a desired range of body magnesium, below which and above which,detrimental effects occur. For example, if body magnesium is too low,then cognitive function may result; however, a diet too high inmagnesium may result in diarrhea. A formulaic approach to determiningoptimum magnesium dosage is more fully detailed in the examplesprovided. In some embodiments, use of the formulas described in theexamples below (and other such methods), will allow a subject tomaintain a dosage regimen which allows for a physiological concentrationas high as possible, without encountering detrimental effects. A desiredbody magnesium status may be defined and/or determined in a variety ofways, including, but not limited to blood magnesium concentration, CSFmagnesium concentration, tissue magnesium concentration, intracellularmagnesium concentration, and red blood cell magnesium concentration.Desired body magnesium status may be applicable for general health aswell as for specific therapeutic applications described herein (e.g.,mild cognitive impairment, AD, depression, osteoporosis, diabetes,etc.). It will be understood that for treatment of different conditions,the optimal body magnesium status may be different to achieve thedesired effects. For instance, by way of example only, it may benecessary to provide a person with a magnesium dosage which willincrease body magnesium concentration by 10% to treat cognitiveimpairment, but a dosage which will increase body magnesiumconcentration by 15% to treat diabetes and/or cardiovascular function.In other words, the compositions described herein can be utilized forthe methods described herein to achieve therapeutically effective bodymagnesium concentrations.

The pharmaceutical compositions can be formulated in slow release orsustained release forms, whereby a relatively consistent level of theactive compound is provided over an extended period. In someembodiments, a magnesium counter-ion composition and/or othertherapeutic agents may be administered jointly or separately by using acontrolled release dosage form. Controlled release within the scope ofthis invention can be taken to mean any one of a number of extendedrelease dosage forms. Extended release dosage forms are described inHeaton et al., U.S. Patent Application Pub. No. US2005/0129762 A1 andEdgren et al. U.S. Patent Application Pub. No. 2007/0128279 A1, whichare herein incorporated by reference. Time-release formulations areknown in the art and are described in Sawada et al. U.S. PatentApplication Pub. No. 2006/0292221 A1, which is herein incorporated byreference. The following terms may be considered to be substantiallyequivalent to controlled release for the purposes of the presentinvention: continuous release, controlled release, delayed release,depot, gradual release, long-term release, programmed release, prolongedrelease, proportionate release, protracted release, repository, retard,slow release, spaced release, sustained release, time coat, timedrelease, delayed action, extended action, layered-time action, longacting, prolonged action, repeated action, slowing acting, sustainedaction, sustained-action medications, and extended release. Furtherdiscussions of these terms may be found in Lesczek Krowczynski,Extended-Release Dosage Forms, 1987 (CRC Press, Inc.). The variouscontrolled release technologies cover a very broad spectrum of drugdosage forms. Controlled release technologies include, but are notlimited to, physical systems and chemical systems.

Use as Excipient

Excipients of the present invention comprise magnesium threonate, withor without augmenting agents. The subject magnesium-counter ioncompound, e.g., magnesium threonate can function as a pharmaceuticallyacceptable excipient. Indeed, compression of pure magnesium threonateyields tablets that retain their shape, are resistant to humidity andhave an acceptable shelf life.

In some embodiments of the invention, magnesium threonate can be pressedinto pill form without an excipient. In other embodiments, magnesiumthreonate may be combined with a pharmaceutically acceptable lubricant,such as magnesium stearate. In stilt other embodiments, magnesiumthreonate may be combined with other ingredients which affect cognitivefunctions and/or general health (e.g., vitamins D and E). In still otherembodiments, a pill, tablet, dragee, lozenge or other acceptablepharmaceutical form may contain magnesium threonate as an excipient andbe combined with another agent of choice, including, but not limited todrugs used to treat AD (e.g., cholinesterase inhibitors Aricept, Exelon,Razadine; glutamate regulators—memantine). One of skill in the art willrecognize that any number of other pharmaceuticals, nutraceuticals,supplements and other components may be added to the dosage forms hereindescribed where magnesium threonate is used as an excipient.

Direct compression tablet manufacturing is preferred for many productsin the pharmaceutical industry. It is a simple process involving lessextensive equipment, operating time and cost. Microcrystalline celluloseis one example of an excipient for direct compression processing.Microcrystalline cellulose has inherently high compactibility due to itsplastic deformation and limited elastic recovery. Microcrystallinecellulose usually provides for good drug dispersion, even ordered mixingwith some drugs and particular grades of microcrystalline cellulose.However, the material flow properties are relatively poor for mostgrades of microcrystalline cellulose. Intermittent and non-uniform flowcan occur as the formulation moves from the hopper to the die on atablet press. This non-uniform flow can lead to drug content variationsin the finished tableted dosage form.

In some embodiments, a wet granulation process will be utilized. Thepopularity of the wet granulation process as compared to the directcompression process is based on at least three potential advantages.First, wet granulation may provide the material to be compacted with amore hydrophilic nature, in order to improve the wetting, disintegrationand dissolution characteristics of some hydrophobic drugs oringredients. Second, the content uniformity and drugsegregation-resistance can be enhanced using a granulation step to lockdrug and excipient components together during blending. Finally, themicrometric characteristics of the component powders can be optimizedprior to compaction, which is often aided by incorporation of apolymeric binder. It is normally considered that this last propertyimbued by wet granulation will yield a significantly more compactableproduct and consequently stronger, more robust tablets.

The present invention is directed in part to a novel use of magnesiumthreonate as a pharmaceutically acceptable excipient.

Depending upon the amount and type of drying, the concentration of themagnesium threonate in the form of a wet cake and any augmenting agentspresent, the compressible particles will have different particle sizes,densities, pH, moisture content, etc. One skilled in the art willappreciate that magnesium threonate may be used in combination withother excipients, including, but not limited to, lactose,microcrystalline cellulose, silicon dioxide, titanium dioxide, stearicacid, starch (corn), sodium starch clycolate, povidone, pregelatinizedstarch, croscarmellose, ethylcellulose, calcium phosphate (dibasic),talc, sucrose, calcium stearate, hydroxy propyl methylcellulose andshellac (and glaze).

Examples of therapeutically active agents for which improveddisintegration results can be obtained include ibuprofen, aldoril, andgemfebrozil, which are relatively high dose (greater than 200 mg/dose)and water-insoluble; verapamil, maxzide, diclofenac and metrolol, whichare moderate-dose drug (25-200 mg/dose) and water-soluble; maproltiline,which is moderate dose (25-200 mg/dose) and water-insoluble; triazolamand minoxidil, which are relatively low dose (less than 25 mg/dose) andwater-soluble. These examples are provided for discussion purposes only,and are intended to demonstrate the broad scope of applicability of theinvention to a wide variety of drugs. It is not meant to limit the scopeof the invention in any way.

Surfactants which may be used in the present invention as acompressibility augmenting agent generally include allpharmaceutically-acceptable surfactants. Suitablepharmaceutically-acceptable anionic surfactants include, for example,those containing carboxylate, sulfonate, and sulfate ions. Thosecontaining carboxylate ions are sometimes referred to as soaps and aregenerally prepared by saponification of natural fatty acid glycerides inalkaline solutions. The most common cations associated with thesesurfactants are sodium, potassium, ammonium and triethanolamine. Thechain length of the fatty acids range from 12 to 18. Although a largenumber of alkyl sulfates are available as surfactants, one particularlypreferred surfactant is sodium lauryl sulfate, which has an HLB value ofabout 40.

In the pharmaceutical arts, sodium lauryl sulfate has been used as anemulsifying agent in amounts of up to about 0.1% by weight of theformulation. Sodium lauryl sulfate is a water-soluble salt, produced asa white or cream powder, crystals, or flakes and is used as a wettingagent and detergent. Also known as dodecyl sodium sulfate, sodium laurylsulfate is actually a mixture of sodium alkyl sulfates consistingchiefly of sodium lauryl sulfate. Sodium lauryl sulfate is also known assulfuric acid monododecyl ester sodium salt. Furthermore, sodium laurylsulfate is readily available from commercial sources such as Sigma orAldrich in both solid form and as a solution. The solubility of sodiumlauryl sulfate is about 1 gm per 10 ml/water. The fatty acids of coconutoil, consisting chiefly of lauric acid, are catalytically hydrogenatedto form the corresponding alcohols. The alcohols are then esterifiedwith sulfuric acid (sulfated) and the resulting mixture of alkylbisulfates (alkyl sulfuric acids) is converted into sodium salts byreacting with alkali under controlled conditions of pH.

Alternative anionic surfactants include docusate salts such as thesodium salt thereof. Other suitable anionic surfactants include, withoutlimitation, alkyl carboxylates, acyl lactylates, alkyl ethercarboxylates, N-acyl sarcosinates, polyvalent alkyl carbonates, N-acylglutamates, fatty acid, polypeptide condensates and sulfuric acidesters.

In other aspects of the invention amphoteric (amphipathic/amphiphilicsurfactants), non-ionic surfactants and/or cationic surfactants areincluded in the coprocessed compositions of the invention. Suitablepharmaceutically-acceptable non-ionic surfactants such as, for example,polyoxyethylene compounds, lecithin, ethoxylated alcohols, ethoxylatedesters, ethoxylated amides, polyoxypropylene compounds, propoxylatedalcohols, ethoxylated/propoxylated block polymers, propoxylated esters,alkanolamides, amine oxides, fatty acid esters of polyhydric alcohols,ethylene glycol esters, diethylene glycol esters, propylene glycolesters, glycerol esters, polyglycerol fatty acid esters, SPAN's (e.g.,sorbitan esters), TWEEN's (i.e., sucrose esters), glucose (dextrose)esters and simethicone.

Other suitable pharmaceutically-acceptable surfactants include acacia,benzalkonium chloride, cholesterol, emulsifying wax, glycerolmonostearate, lanolin alcohols, lecithin, poloxamer, polyoxyethylene,and castor oil derivatives. Those skilled in the art will furtherappreciate that the name and/or method of preparation of the surfactantutilized in the present invention is not determinative of the usefulnessof the product.

Highly polar molecules may also be utilized as the compressibilityaugmenting agent. Such highly polar molecules include certain dyes,particular those which may be capable of binding to the cellulosesurface while thereafter creating a relatively hydrophobic environmentdue to the presence of a hydrophobic portion of the molecule (e.g., ahydrophobic tail) which “points away” from the cellulose surface anddiscourages hydrophilic surface-to-surface cellulose interactions, suchas hydrogen-bonding. Preferably, the dye is one which ispharmaceutically acceptable for inclusion in solid dosage forms.

Examples of suitable dyes include Congo Red (chemical name:3,3′-[[1,1′Biphenyl]-4,4′-diylbis-(azo)]bis[4-amino-1-naphthalenesulfouicacid] disodium salt; FD&C Red No. 40 (also known as “Allura Red”)(chemical name: Disodium salt of6-hydroxy-5[(2-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid);FD&C Yellow No. 5 (common name: tartrazine) (chemical name:5-oxo-1-(p-sulfophenyl)-4-[(p-sulfophenyl)azo]-2-pyrazoline-3-carboxylicacid, trisodium salt); FD&C Yellow No. 6 (common name: Sunset YellowFCF) (chemical name: Disodium salt of1-p-sulphophenylazo-2-naphthol-6-sulfonic acid); Ponceau 4R (chemicalname: Trisodium-2-hydroxy-1-(4-sulfonato-1-naphthylazo)naphthalene-6,8-disulfonate); Brown HT (chemical name: Disodium4,4′-(2,4-dihydroxy-5-hydroxymethyl-3,3-phenylenebisazo)di(napthalene-1-sulfonate)); Brilliant Black BN (Chemical name:Tetrasodium4-acetamido-5-hydroxy-6-[7-sulfonato-4-(4-sulfonatophenylazo)-1-naphthylazo]naphthalene-1,7-disulfonate); Carmoisine (chemical name: Disodium4-hydroxy-3-(4-sulfanato-1-naphythylazo) Naphthalene-1-sulfonate);Amaranth (chemical name: Trisodium2-hydroxy-1-(4-sulfonato-1-naphthylazo) naphthalene-3,6-disulfonate);and mixtures thereof.

Other highly polar molecules which may be utilized as thecompressibility augmenting agent include optional additional activeagents themselves. For example, it is well-known to those skilled in theart that certain classes of pharmaceuticals, such as anti-pyschoticdrugs, are highly polar in nature and may be utilized as acompressibility augmenting agent in accordance with this invention.

The usable concentration range for the selected surfactant depends inpart upon not only its molecular weight but also its degree of foaming,particularly when present in agitated slurries which will be spray driedto form the desired particulate. Thus, in those aspects of the inventionwhere surfactants other than sodium lauryl sulfate are coprocessed withthe magnesium threonate, it is to be understood that the surfactant willbe present in an amount which enhances the compressibility of themagnesium threonate and yet does not have a degree of foaming whichwould substantially inhibit spray drying.

In an embodiment utilizing a spray-drying process, an aqueous dispersionof magnesium threonate and a compressibility augmenting agent (forexample, a surfactant or silicon dioxide) is brought together with asufficient volume of hot air to produce evaporation and drying of theliquid droplets. The highly dispersed slurry is pumpable and capable ofbeing atomized. It is sprayed into a current of warm filtered air, whichsupplies the heat for evaporation and conveys a dried product to acollecting device. The air is then exhausted with the removed moisture.The resultant spray-dried powder particles may be approximatelyspherical in shape and may be relatively uniform in size, therebypossessing excellent flowability. The coprocessed particles are notnecessarily uniform or homogeneous. Other drying techniques such asflash drying, ring drying, micron drying, tray drying, vacuum drying,radio-frequency drying, and possibly microwave drying, may also be used.

Alternatively, all or part of the excipient may be subjected to a wetgranulation with an active ingredient. A representative wet granulationincludes loading the novel excipient particles into a suitablegranulator, such as those available from Baker-Perkins, and granulatingthe particles together with the active ingredient, preferably using anaqueous granulating liquid. In some embodiments, a portion of the totalamount of the novel excipient is wet granulated with the activeingredient, and thereafter the additional portion of the novel excipientis added to the granulate. In yet other embodiments, the additionalportion of the novel excipient to be added to the excipient/activeingredient granulate may be substituted with other excipients commonlyused by those skilled in the art, depending of course upon therequirements of the particular formulation.

In other embodiments of the invention, a further material is added tothe magnesium threonate and/or compressibility augmenting agent. Suchadditional materials include silicon dioxides, non-silicon metal oxides,starches, starch derivatives, surfactants, polyalkylene oxides,cellulose A ethers, celluloses esters, mixtures thereof, and the like.Specific further materials which may be included in the aqueous slurry(and consequently in the resultant agglomerated microcrystallinecellulose excipient) are aluminum oxide, stearic acid, kaolin,polydimethylsiloxane, silica gel, titanium dioxide, diatomaceous earth,corn starch, high amylose corn starch, high amylopectin corn starch,sodium starch glycolate, hydroxylated starch, modified potato starch,mixtures thereof, and the like. These additives may be included indesired amounts which will be apparent to those skilled in the art.

In addition to one or more active ingredients, additionalpharmaceutically acceptable excipients (in the case of pharmaceuticals)or other additives known to those skilled in the art (fornon-pharmaceutical applications) can be added to the novel excipientprior to preparation of the final product. For example, if desired, anygenerally accepted soluble or insoluble inert pharmaceutical filler(diluent) material can be included in the final product (e.g., a soliddosage form). Such inert pharmaceutical filler may comprise amonosaccharide, a disaccharide, a polyhydric alcohol, inorganicphosphates, sulfates or carbonates, and/or mixtures thereof. Examples ofsuitable inert pharmaceutical fillers include sucrose, dextrose,lactose, xylitol, fructose, sorbitol, calcium phosphate, calciumsulfate, calcium carbonate, microcrystalline cellulose, mixturesthereof, and the like.

An effective amount of any generally accepted pharmaceutical lubricant,including the calcium or magnesium soaps may optionally be added to thenovel excipient at the time the medicament is added, or in any eventprior to compression into a solid dosage form. The lubricant maycomprise, for example, magnesium stearate in any amount of about 0.5-3%by weight of the solid dosage form. In embodiments where a surfactant isincluded as part or all of the compressibility augmenting agent, anadditional inclusion lubricant may not be necessary.

The complete mixture, in an amount sufficient to make a uniform batch oftablets, may then subjected to tableting in a conventional productionscale tableting machine at normal compression pressures for thatmachine, e.g., about 1500-10,000 lbs/sq in. The mixture should not becompressed to such a degree that there is subsequent difficulty in itshydration when exposed to gastric fluid.

The average tablet size for round tablets is preferably about 50 mg to500 mg and for capsule-shaped tablets about 200 mg to 2000 mg. However,other formulations prepared in accordance with the present invention maybe suitably shaped for other uses or locations, such as other bodycavities, e.g., periodontal pockets, surgical wounds, vaginally,rectally. It is contemplated that for certain uses, e.g., antacidtablets, vaginal tablets and possibly implants, that the tablet wilt belarger.

The active agent(s) which may be incorporated with the novel excipientdescribed herein into solid dosage forms invention include systemicallyactive therapeutic agents, locally active therapeutic agents,disinfecting agents, chemical impregnants, cleansing agents, deodorants,fragrances, dyes, animal repellents, insect repellents, fertilizingagents, pesticides, herbicides, fungicides, and plant growth stimulants,and the like.

A wide variety of therapeutically active agents can be used inconjunction with the present invention. The therapeutically activeagents (e.g. pharmaceutical agents) which may be used in thecompositions of the present invention include both water soluble andwater insoluble drugs. Examples of such therapeutically active agentsinclude antihistamines (e.g., dimenhydrinate, diphenhydramine,chlorpheniramine and dexchlorpheniramine maleate), analgesics (e.g.,aspirin, codeine, morphine, dihydromorphone, oxycodone, etc.),non-steroidal anti-inflammatory agents (e.g., naproxyn, diclofenac,indomethacin, ibuprofen, sulindac), anti-emetics (e.g., metoclopramide),anti-epileptics (e.g., phenyloin, meprobamate and nitrazepam),vasodilators (e.g., nifedipine, papaverine, diltiazem and nicardirine),anti-tussive agents and expectorants (e.g., codeine phosphate),anti-asthmatics (e.g. theophylline), antacids, anti-spasmodics (e.g.atropine, scopolamine), antidiabetics (e.g., insulin), diuretics (e.g.,ethacrynic acid, bendrofluazide), anti-hypotensives (e.g., propranolol,clonidine), antihypertensives (e.g., clonidine, methyldopa),bronchodilators (e.g., albuterol), steroids (e.g., hydrocortisone,triamcinolone, prednisone), antibiotics (e.g., tetracycline),antihemorrhoidals, hypnotics, psychotropics, antidiarrheals, mucolytics,sedatives, decongestants, laxatives, vitamins, stimulants (includingappetite suppressants such as phenylpropanolamine). The above list isnot meant to be exclusive.

A wide variety of locally active agents can be used in conjunction withthe novel excipient described herein, and include both water soluble andwater insoluble agents. The locally active agent(s) which may beincluded in the controlled release formulation of the present inventionis intended to exert its effect in the environment of use, e.g., theoral cavity, although in some instances the active agent may also havesystemic activity via absorption into the blood via the surroundingmucosa.

The locally active agent(s) include antifungal agents (e.g.,amphotericin B, clotrimazole, nystatin, ketoconazole, miconazol, etc.),antibiotic agents (penicillins, cephalosporins, erythromycin,tetracycline, aminoglycosides, etc.), antiviral agents (e.g, acyclovir,idoxuridine, etc.), breath fresheners (e.g. chlorophyll), antitussiveagents (e.g., dextromethorphan hydrochloride), anti-cariogenic compounds(e.g., metallic salts of fluoride, sodium monofluorophosphate, stannousfluoride, amine fluorides), analgesic agents (e.g., methylsaticylate,salicylic acid, etc.), local anesthetics (e.g., benzocaine), oralantiseptics (e.g., chlorhexidine and salts thereof, hexylresorcinol,dequalinium chloride, cetylpyridinium chloride), anti-inflammatoryagents (e.g., dexamethasone, betamethasone, prednisone, prednisolone,triamcinolone, hydrocortisone, etc.), hormonal agents (oestriol),antiplaque agents (e.g, chlorhexidine and salts thereof, octenidine, andmixtures of thymol, menthol, methysalicylate, eucalyptol), acidityreducing agents (e.g., buffering agents such as potassium phosphatedibasic, calcium carbonate, sodium bicarbonate, sodium and potassiumhydroxide, etc.), and tooth desensitizers (e.g., potassium nitrate).This list is not meant to be exclusive. The solid formulations of theinvention may also include other locally active agents, such asflavorants and sweeteners. Generally any flavoring or food additive suchas those described in Chemicals Used in Food Processing, pub 1274 by theNational Academy of Sciences, pages 63-258 may be used. Generally, thefinal product may include from about 0.1% to about 5% by weightflavorant.

The tablets of the present invention may also contain effective amountsof coloring agents, (e.g., titanium dioxide, F.D. & C. and D. & C. dyes;see the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5, pp.857-884, hereby incorporated by reference), stabilizers, binders, odorcontrolling agents, and preservatives.

Alternatively, the novel excipient can be utilized in other applicationswherein it is not compressed. For example, the granulate can be admixedwith an active ingredient and the mixture then filled into capsules. Thegranulate can further be molded into shapes other than those typicallyassociated with tablets. For example, the granulate together with activeingredient can be molded to “fit” into a particular area in anenvironment of use (e.g., an implant). All such uses would becontemplated by those skilled in the art and are deemed to beencompassed within the scope of the appended claims.

In further embodiments of the invention, more than one compressibilityaugmenting agent is used. Thus, for example, two or more compressibilityenhancing agents are used which provide an effect by differentmechanisms.

EXAMPLES Example 1 Preparation of Magnesium Threonate

Calcium threonate was first prepared from 264 g (1.5 mole) of vitamin C,300 g (3 moles) of calcium carbonate, and 600 mL of 30% by volume H₂0₂,according to the procedure described by Wei et al., J. Org. Chem. 50,3462-3467 (1985). The prepared calcium threonate was redissolved in ˜3 Lwater at ˜90° C. The resulting solution was cooled to ˜50° C. and thenpoured through a 3 inch-diameter column packed with 3 L clean AmberliteIR-120 strongly acidic resin, while the column was continuously elutedwith water. Fractions containing threonic acid having a pH of less thanabout 4.5 were collected. The fractions of threonic acid were combined(˜7 to ˜8 L) and stirred at ˜50 to ˜60° C. Mg(OH)₂ powder was added tothe threonic acid in small portions until the pH reached 7. Theresulting solution was filtered and concentrated by rotary evaporationat ˜50° C. to a final volume of ˜700 to ˜800 mL. The concentratedsolution was cooled to room temperature, filtered to remove any traceamounts of insoluble materials, and then transferred to a 5-L,three-necked, round-bottom flask and mechanically stirred. About 4 L ofmethanol was added to the resulting solution to precipitate out a whitesolid product, magnesium threonate. The solid was collected by suctionfiltration and then dried under high vacuum at 50° C. for 2 days toyield 194 g of magnesium threonate as a white solid. Elemental analysisshowed the material contained one mole of water for each mole ofmagnesium threonate.

Example 2 Taste Comparison

In a double-blind test, each of sixteen human volunteers, 9 males and 7females, varying in age from 20 to 22 years was given one glass of acomposition, Composition 1, comprising skim milk comprising a mixturecomprising 50% by weight of magnesium gluconate, 25% by weight magnesiumlactate, and 25% by weight magnesium citrate, having a 50 mM totalconcentration of elemental magnesium associated with the mixture, andone glass of a composition, Composition 2, comprising skim milk andmagnesium gluconate, having a 50 mM total concentration of elementalmagnesium associated with the magnesium gluconate. Each of thevolunteers was asked to taste the two compositions and state her or hispreference for one or the other or neither. A majority of subjects(87.5%) preferred Composition 1 and a minority of the subjects (12.5%)preferred Composition 2, as graphically depicted in FIG. 1.

Example 3 Enhancement of Magnesium Absorption Rate

Fifty 3-month old, male Sprague Dawley (SD) rats were divided into fivegroups of ten rats. Rats of this age and older are considered adult.Each of the rats was placed in a separate metabolic cage equipped withurine- and feces-collecting wells. All of the rats were maintained in atemperature-controlled room (22° C. to 25° C.) with a dark period from08:00 pm to 08:00 am daily. From day 1 through day 3, each rat was feddaily 15 g of magnesium-free food and de-ionized water. From day 4through day 10, each rat was fed daily 15 g of magnesium-free food andone of five different compositions, Compositions 1-4 and a ControlComposition, containing 12 mM magnesium gluconate in a different medium,depending on its grouping in one of the five groups, Groups 1-4 and aControl Group. The medium was skim milk for Composition 1 and Group 1,milk prepared from powdered milk, by diluting the powdered milk withwater to obtain a composition like that of skim milk, for Composition 2and Group 2, 1% milk cream in water for Composition 3 and Group 3, watercomprising 5 weight percent lactose for Composition 4 and Group 4, andwater for the Control Composition and Control Group. The average volumeof magnesium gluconate solution that was consumed daily was about 35 mL,corresponding to a dosage of elemental magnesium associated with themagnesium-counter ion compound (“elemental magnesium dosage”), here,magnesium gluconate, of about 10 mg/day/rat. From day 11 through day 12,each rat was fed daily 15 g of magnesium-free food and de-ionized water.

From day 4 through day 10, urine from each rat was collected daily. Thecollected urine from each rat was then pooled together and the totalvolume of the pooled urine from each rat was recorded. The pooled urinefrom each rat, in an amount of 500 mL, was analyzed for magnesiumcontent using an inductively coupled plasma-atomic emission spectrometer(ICP-AES). From day 5 to day 11, feces from each rat were collecteddaily. The collected feces from each rat were pooled together and thepooled feces were weighed and homogenized. The pooled feces from eachrat, in an amount of 0.5 g, were analyzed for magnesium content using anICP-AES.

A formula was used to calculate a magnesium absorption rate for eachrat. The formula used was Y=AX−B, wherein X was the average total dailymagnesium intake, Y was the average net daily amount of magnesiumabsorbed, as calculated by X minus the average daily amount of magnesiumexcreted from feces, B was the average daily amount of magnesiumexcreted from feces when the magnesium intake was zero, and the slope Arepresented the magnesium absorption rate. Data points (X, Y) associatedwith each rat in each group often rats, with the exception of the bestpoints and the worst points, were plotted. The value of A, the magnesiumabsorption rate, associated with each of Groups 1-4, and thus with eachof the Compositions 1-4, was then obtained using linear regression. Thevalue of A, the magnesium absorption rate, associated with the ControlGroup, and thus with the Control Composition, was also obtained usinglinear regression, and relabeled as A₀.

A formula was used to calculate a magnesium absorption rate enhancementpercentage for each of Compositions 1-4, based on the magnesiumabsorption rate for each of Compositions 1-4, respectively, relative tothe magnesium absorption rate for the Control Composition. The formulaused was [(A−A₀)/A₀]×100%. The magnesium absorption rates associatedwith each of Compositions 1-4 were all enhanced relative to that for theControl Composition, as graphically depicted in FIG. 2.

Example 4 Enhancement of Magnesium Absorption Rate

A mixture of 50% by weight magnesium gluconate, 25% by weight magnesiumlactate, and 25% by weight magnesium citrate was dissolved in water toprovide a control composition, Control Composition, having a 50 mM totalconcentration of elemental magnesium associated with the mixture. Amixture of 50% by weight magnesium gluconate, 25% by weight magnesiumlactate, and 25% by weight magnesium citrate was dissolved in skim milkto provide a composition, Composition 1, having a 50 mM totalconcentration of elemental magnesium associated with the mixture. Amagnesium absorption rate in rats was determined for each composition inthe manner set forth in Example 3. The magnesium absorption rateassociated with each composition is graphically depicted in FIG. 3. Asshown, the magnesium absorption rate associated with Composition 1 wasgreater than that associated with the Control Composition.

Example 5 Magnesium Absorption Rate Comparison at Different Dosages

A comparison of magnesium absorption rate in rats, as determined in amanner set forth in Example 3, was made for three differentcompositions, each based on a certain magnesium-counter ion compound anda certain medium. Composition 1 was based on magnesium chloride andwater; Composition 2 was based on magnesium gluconate and skim milk; andComposition 3 was based on magnesium gluconate and water comprising 5weight percent lactose. Each of Compositions 1, 2 and 3 was prepared attwo different elemental magnesium concentrations, one providing a 12 mMtotal concentration of elemental magnesium associated with themagnesium-counter ion compound, which corresponded to a total elementalmagnesium intake or dosage of 10 mg/day/rat, and the other providing a36 mM total concentration of elemental magnesium associated with themagnesium-counter ion compound, which corresponded to a total elementalmagnesium intake or dosage of 30 mg/day/rat. A magnesium absorption ratein rats was determined for each composition at each concentration levelin the manner set forth in Example 3. The magnesium absorption rateassociated with each composition at each concentration level isgraphically depicted in FIG. 4. As shown, the magnesium absorption rateassociated with each of Compositions 2 and 3 was higher than thatassociated with Composition 1.

Example 6 Magnesium Absorption Rate Comparison at Different Dosages

A comparison of magnesium absorption rate in rats, as determined in amanner set forth in Example 3, was made for two different compositions,each based on a certain magnesium-counter ion composition and a certainmedium. Composition 1 was based on magnesium chloride and water andComposition 2 was based on magnesium threonate and water. Each ofCompositions 1 and 2 was prepared at two different elemental magnesiumconcentrations, one providing a 12 mM total concentration of elementalmagnesium associated with the magnesium-counter ion compound, whichcorresponded to a total elemental magnesium intake or dosage of 10mg/day/rat, and the other providing a 36 mM total concentration ofelemental magnesium associated with the magnesium-counter ion compound,which corresponded to a total elemental magnesium intake or dosage of 30mg/day/rat. A magnesium absorption rate in rats was determined for eachcomposition at each concentration level in the manner set forth inExample 3. The magnesium absorption rate associated with eachcomposition at each concentration level is graphically depicted in FIG.5. As shown, the magnesium absorption rate associated with Composition 2was greater than that associated with Composition 1 at each of theintake levels, more significantly so at the higher intake level.

Example 7 Measurements of Blood Magnesium Concentration

Twelve 3-month old, male Sprague Dawley (SD) rats were divided into fourgroups of three rats. Each of the rats was placed in a separatemetabolic cage, each of which was maintained in a temperature-controlledroom (22° C. to 25° C.) with a dark period from 08:00 pm to 08:00 amdaily. Each of the rats was fed daily 15 g of normal solid food and adifferent fluid, depending on its grouping in one of the four groups,for three days. A fluid of magnesium chloride in water, Composition 1,was used for Group 1; magnesium threonate in water, Composition 2, forGroup 2; a mixture of 50 weight % magnesium gluconate, 25 weight %magnesium lactate, and 25 weight % magnesium citrate in skim milk,Composition 3, for Group 3; and de-ionized water, Control Composition,for a Control Group. Each of the fluids, other than that for the ControlGroup, was of 35 mM elemental magnesium associated with the subjectmagnesium-counter ion compound, either magnesium chloride for Group 1 ormagnesium threonate for Group 2, or the mixture of magnesium-counter ioncompounds for Group 3. After the three days of feeding as describedabove, about 200 μL of blood was taken from the retrobulbar vein of eachrat. Each of the blood samples was allowed to clot at room temperatureover night, then centrifuged to separate the serum from the clottingfactor, and then analyzed for magnesium concentration using aninductively coupled plasma-mass spectrometer (ICP-MS). The averageconcentration of magnesium in the serum associated with each ofCompositions 1-3 and the Control Composition, respectively, is shown inFIG. 6. As shown, the concentration of magnesium in the serum associatedwith Composition 2 was greater that that associated with Composition 1,Composition 2, and the Control Composition.

Example 8 Measurements of Learning Memory Capacity

A group of 10 mice that were genetically altered to present symptoms ofAlzheimer's disease (AD) were fed an Mg Diet, a diet of normal solidfood and a solution of magnesium threonate and water, for 30 days. Theconcentration of magnesium threonate in the solution was such that theconsumption of a normal amount of the solution corresponded to a totalintake of elemental magnesium associated with the magnesium threonate ofabout 3 mg/day/mouse. Another group, the control group, of 10 mice thatwere genetically altered to present symptoms of AD were fed a ControlDiet, a diet of normal solid food and water, for 30 days.

On the final day of the 30 days of dieting, as described above, eachgroup of mice was trained and tested according to a modified Morriswater maze test (Morris et al., Nature 297, 681-683 (1982)), as nowdescribed. The pool used was a pool of water in a circular metal tank(150 cm in diameter and 50 cm in depth) having a water height of 30 cmand a water temperature that was maintained at ˜22° C. The pool wasplaced in a moderately lit area and surrounded by a black curtain. Anacrylic platform (15 cm in diameter) was placed 2 cm below the surfaceof the water in the middle of one quadrant of the pool, equidistant fromthe center and the edge of the pool. Outside the pool, a cue was placedso as to be visible to a mouse in the maze, allowing a mouse to use itas a landmark for spatial orientation. The cue remained unchangedthroughout the test period.

On the first day of the training and testing period, the water in thepool was transparent, such that the platform was visible. Each mouse wastrained to swim towards the platform and to stand on the platform so asnot to be submerged in the pool. Each mouse underwent a trial, followedby an interval of 1 hour, followed by another trial, and so on, for atotal of 5 trials. In each trial, the subject mouse was placed by handinto the pool of water at a starting or release position that wasrandomly selected from three possible starting positions. The mouseneeded to find the platform so as not to be submerged in the pool. Ifthe mouse found the platform, it was allowed to remain there for 30seconds before it was returned to its home cage. The amount of time themouse took to find the platform, referred to as “escape latency,” wasrecorded for each trial.

On the second day of the training and testing period, a small quantityof milk was added to the water in the pool, such that the pool wasopaque and the platform was no longer visible. Each mouse underwent atrial, followed by an interval of 1 hour, followed by another trial, andso on, for a total of 5 trials. Each trial was as described for thefirst day of the training and testing period. Once again, each subjectmouse placed in the pool needed to find the platform so as not to besubmerged in the pool. The amount of time the mouse took to find theplatform, or escape latency, was recorded and taken as a measure of themouse's short-term spatial memory and learning capacity. A lower escapelatency measurement was associated with a better learning and memorycapacity. If the mouse was unable to find the platform within 90seconds, it was guided to and placed on the platform for 30 seconds,whereupon the trial was ended and the mouse was given a maximum escapelatency score of 90 seconds for the trial.

The two groups of mice underwent further days of training and testing inthe manner described above for the second day of the training andtesting period. An average escape latency associated with the fivetrials was calculated for each group of mice for each of days 2-6 of thetraining and testing period. A graphical representation of these averageescape latency results plotted against the associated day of thetraining and testing period is shown in FIG. 7B. As shown, as the daysin training and testing increased, the average escape latency decreasedfor each group of mice. As also shown, on and after the third day of thetraining and testing period, the mice in the magnesium-fortified dietgroup outperformed the mice in the control group.

Example 9 Measurements of Improvements in Short-Term Spatial MemoryCapacity

Twenty 2-month old, male Sprague Dawley (SD) rats were housed in atemperature-controlled room (22° C. to 25° C.) with a dark period from08:00 pm to 08:00 am daily. Each of the rats was fed a daily diet ofnormal solid food and drinking water on a restricted feeding schedule soas to maintain 85% of its free-feeding weight. Each rat was testedaccording to a version of the T-maze test (Dudchenko, Behav. Neurosci.115, 850-860 (2001)), involving a maze located one meter above the floorof a well-lit laboratory that contained various prominent distalextra-maze cues, which served as landmarks for the rats during the test.Over 7 days before the training and trial period began, each rat washandled and habituated to the maze and to Kellogg's Froot Loop cereal.

In an eight-day training and trial period, each rat was fed a daily dietof normal solid food and drinking water on a restricted feeding scheduleso as to maintain 85% of its free-feeding weight. Each rat underwent atest of one trial, followed by an interval of 10-minutes, followed byanother trial, and so on, for a total of 6 trials in one day. In eachtrial, each rat went through a sample run in the maze, followed by aninterval of 15 seconds, followed by a choice run in the maze. In thesample run, the subject rat was forced to go to the left or to the rightby the presence of a block, according to a pseudorandom sequence (withan equal number of left turns and right turns, and no more than twoconsecutive turns in the same direction). As a reward, Froot Loop cerealwas available in the food well at the end of the run, regardless of thedirection that was taken by virtue of the block. In the choice run, theblock that had been present in the preceding sample run was removed, andthe rat was allowed to choose to go to the left or to the right. As areward, Froot Loop cereal was available in the food well at the end ofthe run, only when the rat had made a “correct choice” by choosing thedirection opposite that taken in the preceding sample run. After 8 daysof the training and trial period, each of the rats attained anasymptotic choice accuracy level, or number of correct choices pernumber of trials, of about 90%, indicating an equal capacity for taskacquisition and working memory.

The rats, once trained and tested as described above, were divided intotwo groups of ten. One group, the control group, was fed a Control Diet,the same daily diet used in the training and trial period, whichincluded normal solid food and drinking water on a restricted feedingschedule so as to maintain 85% of its free-feeding weight. The othergroup was fed an Mg Diet, the same daily diet with the exception that asolution of magnesium threonate (55 mM) in water was used in place ofthe drinking water, on a restricted feeding schedule so as to maintain85% of its free-feeding weight. On average, each of the rats in thelatter group drank about 30 mL of the solution daily, which correspondedto a total intake of elemental magnesium associated with the magnesiumthreonate of about 40 mg/day/mouse, or about 133 mg/kg body weight/day.

On the first day (designated day 0) of the feeding of the two groups, asjust described, each rat underwent a preliminary test of one trial,followed by an interval of 10 minutes, followed by another trial, and soon, for a total of 4 trials in one day. In each trail, each rat wentthrough a sample run in the T-maze described above, followed by aninterval of 15 seconds, followed by a choice run in the maze. In thispreliminary test, the choice accuracy level, or ratio of correct choicesmade, co, to the number of number of trials in the test, no, wasdetermined for each rat. On the fifth day of feeding of the two groups,according to the feeding regime just described, each rat underwentanother test, as described in connection with the preliminary test, toconfirm that the rat still remembered how to complete the trials. On thefollowing day, the sixth day (designated day 6), and on every sixth daythereafter, of feeding according to the same feeding regime, each ratunderwent 4 daily trials, as described above, with the exception that aninterval of 5 minutes was used in place of the interval of 15 seconds.On each day (day i) of such testing, the choice accuracy level, or ratioof correct choices made, c, to the number of trials in the test, n_(i),were determined for each rat. Additionally, a percentage increase in thechoice accuracy level relative to that determined in the preliminarytest was determined for each rat, according to the formula set forthbelow.

$\left( {\frac{{c_{i}/n_{i}} - 0.5}{{c_{0}/n_{0}} - 0.5} - 1} \right) \times 100\; \%$

The percentage increase in the choice accuracy level was taken as ameasure of the rat's short-term working memory and learning capacityimprovement.

An average of the percentage improvement results associated with eachday of testing following the preliminary test was taken for the controlgroup of rats and the other group of rats. A graphical representation ofthese averages versus the number of days on the Mg Diet or the ControlDiet is shown in FIG. 7A. As shown, there was no significant difference(p-value>0.05) in the averages associated with the control group of ratsand the averages associated with the other group of during the firstweek of testing. Thereafter, while there was not a great deal of changein the averages associated with the control group of rats, there was asignificant increase in the averages associated with the latter group ofrats, as demonstrated by the averages associated with day 12 through day24 of being on the Mg Diet, with day 24 showing a 73% difference(p-value<0.05).

Example 10 Effects of Magnesium Supplementation on Recognition Memory

In this example, the effect of magnesium supplementation on recognitionmemory was tested. Three groups of rats were used in theseexperiments: 1) young rats (three months old); aging rats (12-14 monthsold), and; 3) magnesium-treated aging rats (12-14 months old, dietsupplemented with 6 mg/kg MgCl₂ from 8 months of age). We usedexperimentally naive, female, Sprague-Dawley young (2 month old), aging(12-14 month old) and aging (22-24 month old) rats (Charles River) atthe beginning of the behavior experiments. They were housed two per cagewith continuous access to food and water under a 12:12 light-dark cycle,with light onset at 8:00 a.m. Mg2+ levels in CSF in control andMg-treated rats were determined by colorimetric method with xylidyl blue(Thomas, 1998) (Anilytics Incorporated, MD). All experiments involvinganimals were approved by the Massachusetts Institute of Technology's andTsinghua University Committees on Animal Care.

The three groups of rats were tested for recognition memory using anobject recognition test with a single exposure to the object duringtraining. The task is based on the natural tendency of rodents toexplore new objects and tests the animals' memory capacity fordistinguishing novel versus familiar objects. This type of memoryexhibits age-associated decline and correlates with declines in synapticplasticity.

Briefly, the rats were first individually habituated to the personneland then to open-field arena during 2 weeks. The rats were then allowedto explore two identical objects placed into the arena at fixedlocations until they had accumulated 30 of total inspection time (wherethis is defined as active exploration, sniffing or touching the objectwith the nose and/or forepaws) or for a maximum of 20 min. The rat wasreturned to the arena for the retention test and allowed to explore foranother 30 sec. The retention intervals were 10 min and 24 hours.Objects were cleaned thoroughly between trials with 20% ethanol solutionto ensure the absence of olfactory cues. The particular objects for agiven trial were randomly determined, but each object was used for onlyone trial per rat. Memory of the familiar object is associated withincreased exploration of the new object and an exploration index (%correct) is calculated as new object inspection time/30.

As shown in FIG. 8, aging rats displayed a lower novel objectexploration preference at the 10 minute retention interval as comparedto both young rats and aging rats supplemented with magnesium. Thisindicates that aging rats have a learning/memory impairment compared toyoung rats. These results also indicate that magnesium-treated agingrats preferentially explored the novel object to the same extent asyoung rats (P<0.0001).

After 24 hours, all groups lose there ability to distinguish novelversus familiar objects. During the training phase (5 min), both groupsof aging rats showed similar total exploration time for the two objects(P>0.4). This indicates that a difference in exploration time could notaccount for the differences between magnesium-treated and untreatedaging rats.

Example 11 Effects of Liquid and Foodstuff Magnesium Supplementation onMemory Consolidation

In this example, the effect of magnesium supplementation on memoryconsolidation was studied. We used two training sessions separated by 10minutes, before commencing the retention tests (FIG. 9). Training, ratsand magnesium supplementation were carried out essentially as in Example10. Following spaced training, all three groups of rats (young, aging,and magnesium-supplemented aging) showed a similar preference for thenovel object at the 10 min retention interval, suggesting that the agingrats were still capable of performing the task with multiple trainingtrials. However, at the 24-hour retention interval, the untreated agingrats showed no preference for the novel object (P<0.005), whilemagnesium-treated aging rats retained a high level of preference. Theseresults demonstrate the effectiveness of magnesium treatment in theprevention of age-dependent recognition memory decline in aging rats.

Enhancement of short term memory for rats receiving magnesiumsupplementation was also determined using lactose-supplementedmagnesium. For these experiments, the magnesium mixture described above(magnesium gluconate, magnesium lactate and magnesium citrate) and 5%lactose were added to the drinking water of rats being tested (40 mgmagnesium/day). Following one week of treatment, short-term memory wasdetermined using the novel object recognition test, essentially asdescribed in Example 10. This experiment mimics the results of magnesiumsupplementation in milk as it was determined that lactose is the uptakeenhancing factor in milk. Results are shown in FIG. 11. These resultsshow that rats receiving magnesium supplementation spend more timeexamining the novel object, suggesting an improvement of short-termmemory.

In a similar experiment, rats are fed magnesium-threonate supplementedchocolate. The rats are given unlimited access to their normal diet.Water is available at all times, except during brief testing periods.The rats are approximately 6 months old at the beginning of theexperiment. A 45-mg pellet dispenser (ENV-203) is placed behind eachfood trough. Rats are provided access to magnesium compositionsupplemented chocolate pellets such that when consumed, the chocolatepellets will provide 20-40 mg of elemental magnesium per day.

Example 12 Effects of Magnesium Supplementation on Spatial WorkingMemory

Three groups of animals (young, aging, and magnesium-treated aging rats)were used. Animals and diets were as described in Example 10. Spatialworking memory was assessed using a T-maze non-matching-to-place task.Briefly, rats were maintained on a restricted feeding schedule at 85% oftheir free-feeding weight. Spatial working memory was first assessed onan elevated T-maze. The maze was located 1 m above the floor in a welllit laboratory that contained various prominent distal extra-maze cues.The rats were handled and habituated to the maze for 10 days, and toFroot Loop® cereal over several days before the test. Each trialconsisted of a sample run and a choice run, with delay intervals of 15 sduring the training and the pattern completion tasks. On the sample ran,the rats were forced either left or right by the presence of the block,according to a pseudorandom sequence (with equal numbers of left andright turns per session, and with no more than two consecutive turns inthe same direction). A cereal reward was available in the food well atthe end of the arm. The block was then removed, and the rat was alloweda free choice of either arm. The animal was rewarded for choosing thepreviously unvisited arm. Rats were run one trial at a time with aninter-trial interval of 10 min. Each daily session consisted of 6trials.

The rats were tested for 10 consecutive days on a rewarded forced-choicealternation task. The percentage of correct choices (alternations) wasrecorded for each daily session. In our experiments, the animals likelyused a spatial strategy since, when the maze was rotated 180°, theanimals went to the arm predicted by allocentric rather than egocentricinformation (data not shown). Aging rats displayed impaired learning innon-matching-to-place task as compared to young rats (FIG. 10, leftpanel, 15 sec delay). Magnesium-treated aging rats performedsignificantly better from their first trials (p<0.05). After 8 days oftraining, all three groups attained an asymptotic choice accuracy levelof 94%, suggesting an equal capacity for task acquisition. Then, spatialworking memory was tested by a gradual increase of the delay between thesample and the choice trials (FIG. 10, right panel). No difference wasfound between young and aging rats across different delays (p>0.05),while magnesium-treatment significantly enhanced the performance of theaging rats at 2 and 5 min delays (p<0.05). Thus, although spatialworking memory evaluated by T-maze did not decline with aging,magnesium-treated aging rats have enhanced spatial working andshort-term memory.

Example 13 Effects of Magnesium Threonate on Learning and Memory of AgedRats

To test whether intake of magnesium threonate leads to the improvementof working memory, learning and memory of aged (22-24 month old) ratswith profound memory deficiency was examined. Twenty-four aged rats weretrained to perform the elevated T maze (described in the previousexample) for 10 days. Their working memory was evaluated by choiceaccuracy between the sample and choice trials with increasing delay. Toensure similar averaged working memory between control andmagnesium-treated groups before the start of magnesium treatment,animals were randomly assigned for two groups in the end of training.Then, drinking water of rats in magnesium-treated group was supplementedwith magnesium threonate (100 mg/kg/day). The effect of magnesiumtreatment on the rats' working memory was evaluated every six days (FIG.7C).

The choice accuracy continuously declined in the control group duringthe repeated sampling. However, 12 days after beginning magnesiumthreonate treatment, choice accuracy associated with longer delays beganto increase in the magnesium-treated group and reached to its peak onthe day 24 (P<0.05, N=12). These data suggest that magnesium threonateimproves working memory.

To determine whether Mg treatment triggers reversal of memory decline orgeneral memory enhancement, we tested the efficiency of Mg treatment inyoung rats (2 month old). Using similar experimental procedures as thoseused for aged rats, the data demonstrate that magnesium threonatesignificantly enhanced the working memory of young rats at the 5 mindelay time point compared to a control group of untreated rats withstable performance (FIG. 7C). Therefore, increasing magnesiumconsumption generally enhances working memory of young and aged rats.

Twenty 2-month old, male Sprague Dawley (SD) rats were housed in atemperature-controlled room (22° C. to 25° C.) with a dark period from08:00 pm to 08:00 am daily. Each of the rats was fed a daily diet ofnormal solid food and drinking water on a restricted feeding schedule soas to maintain 85% of its free-feeding weight. Each rat was testedaccording to a version of the T-maze test (Dudchenko, Behav Neurosci.115, 850-860 (2001)), involving a maze located one meter above the floorof a well-lit laboratory that contained various prominent distalextra-maze cues, which served as landmarks for the rats during the test.Over 7 days before the training and trial period began, each rat washandled and habituated to the maze and to Kellogg's Froot Loop cereal.

In an eight-day training and trial period, each rat was fed a daily dietof normal solid food and drinking water on a restricted feeding scheduleso as to maintain 85% of its free-feeding weight. Each rat underwent atest of one trial, followed by an interval of 10-minutes, followed byanother trial, and so on, for six trials in one day. In each trial, eachrat went through a sample run in the maze, followed by an interval of 15seconds, followed by a choice run in the maze. In the sample run, thesubject rat was forced to go to the left or to the right by the presenceof a block, according to a pseudorandom sequence (with an equal numberof left turns and right turns, and no more than two consecutive turns inthe same direction). As a reward, Froot Loop cereal was available in thefood well at the end of the run, regardless of the direction that wastaken by virtue of the block. In the choice run, the block that had beenpresent in the preceding sample run was removed, and the rat was allowedto choose to go to the left or to the right. As a reward, Froot Loopcereal was available in the food well at the end of the run, only whenthe rat had made a “correct choice” by choosing the direction oppositethat taken in the preceding sample run. After 8 days of the training andtrial period, each of the rats attained an asymptotic choice accuracylevel, or number of correct choices per number of trials, of about 90%,indicating an equal capacity for task acquisition and working memory.

The rats, once trained and tested as described above, were divided intotwo groups of ten. One group, the control group, was fed a Control Diet,the same daily diet used in the training and trial period, whichincluded normal solid food and drinking water on a restricted feedingschedule so as to maintain 85% of its free-feeding weight. The othergroup was fed an Mg Diet, the same daily diet with the exception that asolution of magnesium threonate (55 mM) in water was used in place ofthe drinking water, on a restricted feeding schedule so as to maintain85% of its free-feeding weight. On average, each of the rats in thelatter group drank about 30 ml of the solution daily, which correspondedto a total intake of elemental magnesium associated with the magnesiumthreonate of about 40 mg/day/mouse, or about 133 mg/kg body weight/day.

On the first day (designated day 0) of the feeding of the two groups, asjust described, each rat underwent a preliminary test of one trial,followed by an interval of 10 minutes, followed by another trial, and soon, for a total of four trials in one day. In each trail, each rat wentthrough a sample run in the T-maze described above, followed by aninterval of 5 minutes, followed by a choice run in the maze. On thefifth day of feeding of the two groups, according to the feeding regimejust described, each rat underwent another test, as described inconnection with the preliminary test, to confirm that the rat stillremembered how to complete the trials. On the following day, the sixthday (designated day 6), and on every sixth day thereafter, of feedingaccording to the same feeding regime, each rat underwent 4 daily trials,as described above. On each day (day i) of such testing, the choiceaccuracy level, or ratio of correct choices made to the number of trialsin the test, were determined for each rat.

An average of the percentage choice accuracy associated with each day oftesting following the preliminary test was taken for the control groupof rats and the Mg treated group of rats. The difference between twogroups versus the number of days on the magnesium Diet or the ControlDiet is shown in FIG. 7A. As shown, there was a significant increase inthe averages associated with the magnesium treated group of rats,starting around day 12 through day 24 of being on the Mg Diet, with day24 showing a 25% increase (p-value<0.05). Similar phenomena occur inaged animal (17 month old) under magnesium treatment (FIG. 7C).

Example 14 Effects of Magnesium Threonate on Working Memory

Having demonstrated the enhancement of working memory by magnesiumtreatment, further experiments were conducted to determine whethermagnesium threonate led to the improvement of long-term memory in youngand aged rats using the Morris water maze. For these experiments,drinking water was supplemented with magnesium threonate (100 mg/kg/day)in the magnesium-treated groups. Briefly, the Morris water maze task wasused to study spatial learning and memory after distinct difference inT-maze working memory test was observed, and the method is as describedpreviously, with minor modifications. The pool was a circular metaltank, 150 cm in diameter, 50 cm deep, filled to a height of 30 cm withwater. Water temperature was maintained at ˜22° C. An acrylic platform(15 cm in diameter) was placed inside the pool, its upper surface 2 cmbelow the surface of the water, so that a rat inside the pool would beunable to locate it visually. The pool was set in a moderately lit,circular enclosure made with black curtain, in which there were severalcues (two for young rats and four for old rats) with different sharp andcolor external to the maze. These were visible from within the pool andcould be used by the rat for spatial orientation. These cues remainedunchanged throughout the testing period.

The young rats undergo 8 trials training with an inter-trial interval of1 hour for one day. For old rats, the training session was split intotwo days, 5 trials for day1 and 3 trials for day2, and the inter-trialinterval is also 1 hour. Each rat was placed into the water by hand, sothat it faced the wall of the pool, at one of three starting positions.The sequence of these positions was randomly selected. The platform wasset in the middle of one quadrant, equidistant from the center and theedge of the pool. If the rat found the platform, it was allowed toremain there for 30 s and was then returned to its home cage. If the ratwas unable to find the platform within 90 s, it was guided to and placedon the platform for 30 s, the trial was terminated and the maximum scoreof 90 s was given. In each trial the goal latency to the hidden platformwas recorded using a video system, Ethovision (Nadolus).

The probe trial (also the memory retention test) was carried out 1 hour(first probe trial) and 24 hours (second probe trial) after the lasttrial of the training session. In the probe trial, the platform wasremoved and each rat was put into the pool for 30 s. The total timespent in the target quadrant (where the platform had been located duringthe training trials), as well as the swimming speed, was measured usingthe same video system.

After finishing the probe trial, the rats receive partial cue test toaccess their ability to retrieve memories on the basis of incompleteinformation. First rats received re-training in which the platform wasput back in the same location compared with the training session. Afterthe rats remembered the location of platform, the cues were adjustedthat only one cue was remained in the experiment system, and the escapelatency of rats in this circumstance was recorded. Then, a full-cue testwas carried and the escape latency was recorded.

For these experiments, rats and diets were essentially the same asdescribed in Example 13. During the training period, the performance ofcontrol and magnesium threonate-treated rats gradually improved in bothyoung and aged groups (FIG. 12). However, magnesium-treated rats learnedfaster than control rats (ANOVA test, young: F (7, 215)=17.07, p<0.001,n=15; aged: F(7,215)=17.11, p<0.001, n=15).

In the probe tests performed 1 hour after the end of the training (whenthe platform was removed and the rats were allowed to search for 60seconds), all four groups of rats (young, magnesium-treated young, aged,magnesium-treated aged) showed preference for the training quadrant(young, FIG. 13, left panel, p<0.001; aged, FIG. 13, right panel,p<0.001), suggesting that young and aged groups are able to equallymemorize the location of the platform.

To test the rats' long-term spatial memory, the probe tests were delayed24 hours after the training. The control rats in both young and agedgroups lost their preference for the training quadrant (p>0.25), whilemagnesium-treated young (FIG. 13, left panel) and aged (FIG. 13, rightpanel) rats retained their quadrant preference (young rats: p<0.001;aged rats: p<0.01). Vision and locomotor functions were equallyefficient in both group of rats, judging by swimming speed and latencyof escape to a visible platform (young rats: p=0.83; aged rats: p 0.84).Thus, these results demonstrate that magnesium threonate significantlyenhances hippocampus-dependent learning and memory in both young andaged rats.

Another crucial function of biological memory systems exhibitingprofound decline during aging is pattern completion—the ability toretrieve memories on the basis of incomplete information. We studied thedependence of spatial memory recall on the integrity of distal cuesduring water maze test. The pattern completion experiments wereperformed with aged rats that underwent the training period in watermaze (FIG. 14). Magnesium-treated aged rats performed better underpartial-cue conditions than control aged rats in water maze (FIG. 14).Magnesium-treated rats had similar escape latency at full-cue and atpartial-cue conditions in water maze (p=0.75), whereas the escapelatency of control aged rats increased significantly under partial-cuecondition (FIG. 14, p<0.05). These results indicate that magnesiumthreonate treatment is effective for improving memory recall in agedrats.

Example 15 Effects of Magnesium Threonate in a Mouse Alzheimer's Disease(AD) Model

In this example, the potential for treatment of AD with magnesiumthreonate was analyzed. For these experiments, [insert mouse strainparameters—include control, 6 month/13 month,—here] were utilized. ADmice were given 3 mg/per day of elementary magnesium in form ofmagnesium threonate (MgT). For these experiments, mice were tested usingthe Morris water maze test, essentially as described in the previousexample. Results are shown in FIG. 15.

During the training period, the performance of control, AD and magnesiumthreonate-treated AD mice gradually improved in young mice (FIG. 15,panel A). However, young AD mice treated with MgT showed a similarlearning progression to control mice. Aged AD mice showed no improvementduring the training period, however, control and MgT-treated AD mice didshow improvement during the training period (FIG. 15, panel C). Thisdemonstrates that MgT is effective in counteracting the effects of ADduring the learning process in both young and old mice.

Young control mice, young MgT-treated AD mice, aged control mice andaged MgT-treated AD mice showed preference for the training quadrant(FIG. 15, panels B and D). These results show several things. First, theresults suggest that young and aged groups are able to equally memorizethe location of the platform. Second, the results demonstrate that MgTtreatment is able to counteract the effects of AD on long-term spatialmemory.

Example 16 Comparison of Magnesium Threonate with Anti-AD Drugs

Having demonstrated the effectiveness of MgT treatment in counteractingthe effects of AD, a comparison with other anti-AD drugs was performed.In this example, the effectiveness of magnesium threonate in treating ADwas compared to the effectiveness of other anti-AD drugs. For theseexperiments, the mice (aged 13 months) and magnesium threonatesupplementation were essentially as described in Example 14. Two knownanti-AD drugs named aricept and memantine were administered separatelyto the mice. For these experiments, mice were tested for effects onmemory and learning using the Morris water maze test, essentially asdescribed in the previous example. Results are shown in FIG. 16.

Initially, there was little difference between WT and AD mice receivingtreatment with any of the test compounds. However, AD mice treated withMgT and memantine showed similar effects, both being better at reducingthe effects of AD on learning capacity than aricept (FIG. 16, panels Aand B).

Example 17 Correlation Between Short-Term Memory and Magnesium Intake inAged Rats

In this example, the effect of magnesium supplementation on recognitionmemory was tested in aging rats (12-14 months old). We usedexperimentally naive, male, Sprague-Dawley rats (Charles River) at thebeginning of the behavior experiments. They were housed two per cagewith continuous access to food and water under a 12:12 light-dark cycle,with light onset at 8:00 a.m. The total magnesium intake/rat wasdetermined by adding the sum of magnesium from food and magnesiumsupplement (Mg threonate) in their drinking water.

The rats were tested for recognition memory using an object recognitiontest with a single exposure to the object during training. The task isbased on the natural tendency of rodents to explore new objects andtests the animals' memory capacity for distinguishing novel versusfamiliar objects. This type of memory exhibits age-associated declineand correlates with declines in synaptic plasticity.

Briefly, the rats were first individually habituated to the personneland then to open-field arena during 2 weeks. The rats were then allowedto explore two identical objects placed into the arena at fixedlocations until they had accumulated 30 of total inspection time (wherethis is defined as active exploration, sniffing or touching the objectwith the nose and for forepaws) or for a maximum of 20 min. The rat wasreturned to the arena for the retention test and allowed to explore foranother 30 sec. The retention intervals were 10 min for short-termmemory test. Objects were cleaned thoroughly between trials with 20%ethanol solution to ensure the absence of olfactory cues. The particularobjects for a given trial were randomly determined, but each object wasused for only one trial per rat. Memory of the familiar object isassociated with increased exploration of the new object.

As shown in FIG. 19, in comparison with rat in control group (denoted byopen squares; n=10) the animal with Mg compound treatment (denoted byfilled squares; n=9) show higher exploration preference to novel object,suggesting the improvement of their short-term memory. More importantly,the degree of improvement is strongly correlated with the amount of Mgsupplement they intake (p<0.01). This experiment clearly shows thatanimals with higher total magnesium intake have better short-termmemory.

Example 18 Correlation Between Short-Term Memory and Plasma MagnesiumConcentration in AD Mice

In this example, the correlation between short-term memory and plasmamagnesium concentration in AD mice was determined. The novel objectrecognition test was used to evaluate the short-term memory of AD micereceiving magnesium treatment. The experimental procedure is similar towhat described in Example 16 except that four objects were used (threeold and one new) in each test. The exploration preference to novelobject in AD mice is linearly correlated with their plasma Magnesiumvalues (n=11, p<0.05). Results are shown in FIG. 20.

The significance of Examples 16 and 17 is that for the first time weestablished that cognitive function improvement is linearly correlatedto magnesium intake, which is, in turn, linearly correlated to bloodmagnesium level. These results are unexpected as it was equallyreasonable to expect that only when magnesium intake or blood magnesiumlevels reach a certain threshold level can cognitive function beimproved. Furthermore, without these discoveries, one of ordinary skillwould not know to what extent an animal's cognitive function can beimproved. Our data suggest that magnesium intake should be as high aspractical as long as the intake does not cause diarrhea and the bloodmagnesium level does not exceed the upper limit of the normal bloodmagnesium distribution range (i.e., induce hypermagnesia effects). Thus,we here present the foundations for determining the optimal dosage rangeand regimen for any suitable magnesium compound which maintains bloodmagnesium concentrations at the high end of the normal blood magnesiumdistribution range for a given animal species.

Example 19 Correlation Between Physical Motility of AD Mice in aDose-Dependent Fashion

In this example, we demonstrate the correlation between physicalmotility of AD mice in a dose-dependent fashion. The movement of miceduring water maze test (similar to the test described in Example 8above) was monitored with video camera. The swimming speed of each miceis calculated from off-analysis. Results are shown in FIG. 21. As can beseen from these results, magnesium treatment of AD mice following 7months of treatment (FIG. 21, left panel) and 15 months of treatment(FIG. 21, right panel) resulted in greatly increased mobility during thewater maze test.

Example 20 Sustained Improvement of Learning and Memory Functions of ADMice Receiving Magnesium Supplementation

In this example, the ability of magnesium supplementation to sustainimprovement of learning and memory functions of AD mice. A group of 10mice that were genetically altered to present symptoms of Alzheimer'sdisease (AD) were fed a Magnesium Diet (a diet of normal solid food anda solution of magnesium threonate and water). The concentration ofmagnesium threonate in the solution was such that the consumption of anormal amount of the solution corresponded to a total intake ofelemental magnesium associated with the magnesium threonate of about 3mg/day/mouse. Another group, the control group, of 10 mice that weregenetically altered to present symptoms of AD was fed a Control Diet, (adiet of no-1solid food and water).

On the final day of the 60 days on the described diets, each group ofmice was trained and tested according to a modified Morris water mazetest (Morris et al., Nature 297, 681-683 (1982)), as now described. Thepool used was a pool of water in a circular metal tank (150 cm indiameter and 50 cm in depth) having a water height of 30 cm and a watertemperature that was maintained at 22° C. The pool was placed in amoderately lit area and surrounded by a black curtain. An acrylicplatform (15 cm in diameter) was placed 2 cm below the surface of thewater in the middle of one quadrant of the pool, equidistant from thecenter and the edge of the pool. Outside the pool, cues were placed soas to be visible to a mouse in the maze, allowing a mouse to use it as alandmark for spatial orientation. The cues remained unchanged throughoutthe test period.

On the first day of the training and testing period, the water in thepool was transparent, such that the platform was visible. Each mouse wastrained to swim towards the platform and to stand on the platform so asnot to be submerged in the pool. Each mouse underwent a trial, followedby an interval of 1 hour, followed by another trial, and so on, for fivetrials. In each trial, the subject mouse was placed by hand into thepool of water at a starting or release position that was randomlyselected from three possible starting positions. The mouse needed tofind the platform so as not to be submerged in the pool. If the mousefound the platform, it was allowed to remain there for 30 seconds beforeit was returned to its home cage. The amount of time the mouse took tofind the platform, referred to as “escape latency,” was recorded foreach trial. On the second day of the training and testing period, asmall quantity of milk was added to the water in the pool, such that thepool was opaque and the platform was no longer visible. Each mouseunderwent a trial, followed by an interval of 1 hour, followed byanother trial, and so on, for five trials. Each trial was as describedfor the first day of the training and testing period. Once again, eachsubject mouse placed in the pool needed to find the platform so as notto be submerged in the pool. The amount of time the mouse took to findthe platform, or escape latency, was recorded and taken as a measure ofthe mouse's short-term spatial memory and learning capacity. A lowerescape latency measurement was associated with a better learning andmemory capacity. If the mouse was unable to find the platform within 90seconds, it was guided to and placed on the platform for 30 seconds,whereupon the trial was ended and the mouse was given a maximum escapelatency score of 90 seconds for the trial.

The two groups of mice underwent further days of training and testing inthe manner described above for the second day of the training andtesting period. An average escape latency associated with the fivetrials was calculated for each group of mice for each of days 2-6 of thetraining and testing period. A graphical representation of these averageescape latency results plotted against the associated day of thetraining and testing period is shown in FIG. 15 (panels A and C). Asshown, as the days in training and testing increased, the average escapelatency decreased for each group of mice. As also shown, on and afterthe third day of the training and testing period, the mice in themagnesium-fortified diet group outperformed the mice in the controlgroup.

To check the long effects of magnesium compound treatment, the AD micein magnesium treated were under Magnesium diet continuously. Thelearning capabilities of three of mice were evaluated using the watermaze test 10 months after beginning the diet. AD mice fail to find thehidden platform completely, while wild type mice and AD mice undermagnesium treatment can still find the location of hidden platformquickly (data not shown). These results show that magnesium treatment isstill effective after long-term treatment.

Finally, even after 15 month of magnesium treatment (via the dietsdescribed above), the short-term memory of AD mice (measured using anovel object recognition test as described above) were still as good asthe wild type control mice, while the AD mice without magnesiumtreatment have very poor short-term memory (data not shown).

Example 21 Ameliorative Effects of Magnesium Supplementation onDepression

In this example, a forced swimming test (FST) was used to evaluateanti-depression effects of Magnesium compound. FST is the most widelyused tool for assessing antidepressant activity preclinically. The testfollows the method described by Porsolt et al., Nature, 266: 730-2(1977) with a little modification to increase its sensitivity (Cryan etal., Trends Pharmacol. Sci., 23:23845 (2002)). Animals were individuallyplaced into glass cylinders (50 cm height; 20 cm diameter) containing 40cm of water at 22° C. After 15 min, they were transferred to a 30° C.drying environment for 30 min (the pre-test phase). The animals werereturned to the cylinder 24 h later for 5 min (the test phase), and thissession was recorded with a video camera. Fresh water was used for eachrat and the cylinder was cleaned. Experiments were performed between10:00 a.m. and 3:00 p.m. Observation of the videotapes was performed byan experimenter unaware of the treatment received by the animals andimmobility time measured. A rat was considered immobile when floatingand making only the necessary movements to keep its nostrils above thewater surface. Additionally, animals behavior during test phase wasdivided into swimming, climbing and immobility during 5 sec intervals,then data were analyzed as described (Cryan et al., 2002).

A significant reduction in immobility of animals treated with magnesiumthreonate in comparison with controls was observed after chronicmagnesium threonate consumption. Interestingly, the immobility time ofmagnesium threonate-treated animals significantly correlated withmagnesium threonate intake (FIG. 22). These results show that, like theeffect on cognitive function, magnesium has antidepressant effect alsoin a dose-pendent fashion. The result suggests that the optimal dosagerange and regimen for a magnesium compound to enhance cognitive functionare equally applicable to utilization of magnesium as an antidepressant.

Example 22 Increased Lifespan of Drosophila Receiving MagnesiumThreonate

To examine the effect of magnesium on an animal's lifespan, two standardlaboratory inbred strains of Drosophila, 2 U and Canton S(CS) wild-typeflies, were fed magnesium threonate (MgT). The flies were reared inbottles or vials maintained at 25° C. and 65% humidity on a 12-hourlight/12-hour dark cycle. The 2 U line was reared in Cold SpringHarbor's standard laboratory fly medium. The CS line was reared instandard density culture on standard laboratory fly medium. TheMagnesium-supplemented media were prepared by adding MgT to vigorouslystirred normal molten media at 70° C. The final concentration of MgT infood for the 2 U line was 80, 160, 240 and 400 ug/g, respectively, whilethe final concentration of compound in food for the CS line was 100,200, 300 and 500 ug/g, respectively. The flies were initially reared in30 mL-sized transparent plastic bottles containing 4 mL food media.Newborn flies on the day of eclosion were transferred to mediumcontaining different concentration of MgT for 2 days for mating. Afterthat, male and female flies were transferred to vials (20/vial) underlight CO2 anesthesia. There were around 200 flies in each treatment.Flies were transferred to vials containing fresh medium every 2 days anddeaths were scored daily. Data were plotted either as survival rate vs.time (FIG. 23) or as percent lifespan change vs. fold in the amount ofMagnesium increase in food (FIG. 24) from multiple trials.

The results suggest that the benefit of magnesium supplementation is notlimited to cognitive function—it improves the overall health of theanimal. It also suggests that there exists an optimal magnesium dosagerange. Too high a dosage or a body magnesium level may diminish thebenefit or even cause harm. Thus, this data also provides furthersupport for establishing the optimal range of supplementation thatyields health benefits.

Example 23 Measuring Plasma, Serum or Urine Magnesium Concentration

In this example, we develop a new method for determining physiologicalconcentrations of magnesium. The data discussed above demonstrates thata relatively high body magnesium level is important for maximal healthbenefit, but too high a magnesium level may be harmful. Therefore, it isdesirable for an individual to take the right amount of a magnesiumsupplement so that the desired body magnesium level is achieved. To dothis, two requirements need to be met. The first is a reliable way ofassessing body magnesium level. The second is an efficient andcontrollable magnesium supplementation technique. Here we disclose themethod derived from the data we have collected, which provided theinformation allowing us to achieve both requirements.

We have discovered that following a meal, the blood magnesium level(such as [Mg]_(plasma)) rises rapidly, reaching a peak and then fallingback to a baseline level. It is the baseline level blood magnesiumconcentration (‘basal [Mg]”) that is indicative of body magnesiumstatus. The magnesium concentration at or near the peak is highlyvariable, depending on the amount and type of food ingested. Thus, ifthe blood magnesium is measured following a meal, the value is likely tobe too high and variable in nature. Most clinical guidelines formeasuring blood magnesium state that it is not necessary to fast beforea blood sample is taken. This may at least partly explain the widedisparity in the reported normal ranges of blood magnesium concentrationfor both healthy and unhealthy subjects.

The significance of our finding is two fold. First, basal bloodmagnesium concentration measured after 12 hour fasting is morereflective of the true body magnesium status. Second, magnesiumsupplementation should be preferably taken between meals, and mostpreferably taken before bedtime. The supplement is preferably a liquidform, or more preferably a slow-release solid form. The underlyingreason is that when blood magnesium concentration peaks, most magnesiumis excreted in the urine via the kidneys. Thus, it is preferable tostagger the meal times and supplementation times so that a moresustained blood magnesium concentration is achieved, allowing more timefor blood magnesium to distribute to tissues. Even more preferably, themagnesium supplementation is taken at bedtime

Body magnesium status may be assessed in one of many ways or in acombination of several ways. Other body Magnesium status indicators anddetection methods include the following: 1) intracellular ionizedmagnesium in red blood cells; 2) bone magnesium content; 3) magnesiumconcentration in the cerebrospinal fluid; 4) sublingual magnesium assay(e.g., use of the ‘Exatest’ is a test used, for example, during cardiacsurgery to determine cellular magnesium levels.); 5) intracellular freemagnesium; and 6) nuclear magnetic resonance (NMR) spectroscopy. SeeBuchli and Duc, Magn. Reson. Med. 32:47-52 (1994).

For this example, Calmagite, a Mg²⁺ chelating dye, was used formeasuring [Mg]_(plasma) and [Mg]_(urine) in an alkaline (pH>11) solution(See, e.g., Khayam-Bashi, et al., Clin. Chem. 23: 289-91 (1977);Abernethy and Fowler, Clin. Chem. 28: 520-22 (1982); and Liedtke andKroon, Clin. Chem. 30: 1801-4 (1984)). Upon binding to Mg²⁺, the bluecolored dye Calmagite forms a pink colored Calmagite-Mg²⁺ complex withan absorption maximum at ˜520 nm. According to Lambert-Beer's law, Mg²⁺concentration between 0˜2.5 mM has a linear correlation with absorbancevalue at 520 nm. Thus, [Mg²⁺] in a sample can be obtained from theabsorbance at 520 nm and a standard curve.

For all [Mg²⁺] measurements through out this study, a Calmagite workingsolution containing EGTA, Strontium chloride and AMP was preparedaccording to the above cited references. The purpose of adding EGTA,strontium chloride and AMP was to remove the interference of calcium andiron. A standard curve was first generated by using a series of eitherMgSO₄ or MgCl₂ solutions with known concentrations (standard solutions).A small volume (50 uL) of a standard solution was added to 2 mL dyeworking solution in a quartz cuvvete. Following a brief incubation, theabsorbance of the solution at 520 nm was measured to give A₁ using aBeckman Uv/Vis 530 spectrophotometer. Subsequently, 5 uL of 150 nm EDTAsolution was added to the above solution, followed by 1 minute ofincubation to break up the Magnesium-Calmagite complex. The solution wasincubated until the absorbance at 520 mm became stable. This stableabsorbance value, A₂, was the background absorbance. A standard curvewas generated by plotting (A₁-A₂) vs. [Mg²⁺]_(standard). Plasma or urinesamples were measured according to the same procedure used forgenerating the standard curve except that the urine samples werediluted, if necessary, to below 2.5 mM. Magnesium concentrations of thesamples were then obtained from the (A₁-A₂) values and standard curve.The bioavailability of three magnesium compositions, magnesiumdiglycinate, magnesium gluconate and magnesium gluconate in milk (at 0.8mg/mL), were compared in three healthy male volunteers. Before magnesiumsupplementation began, urine samples of the volunteers were collectedfor 2 days. Then, the volunteers were asked to take either of the threemagnesium compositions at the amount of 200 mg magnesium each time twiceper day for 2 days, during which the urine samples were collected. Allurine samples were analyzed for their magnesium contents using the dyemethod as described in above. Cumulative urinary magnesium excretion wasused to determine the bioavailability (magnesium absorption rate) ofeach magnesium composition according to the reported procedure using theformula below (Drenick, E. J., et al., J. Clin. Endocrinol Metab, 1969.29(10): p. 1341-8; Lim & Jacob, Metabolism, 1972. 21(11): p. 1045-51):

k _(x)=(Mg _(u) ² −Mg _(u) ¹)/dosage

where k_(x) is the magnesium absorption rate; Mg_(u) ² is the amount of2-day urine magnesium with magnesium supplementation; Mg_(u) ¹ is theamount of 2-day urine magnesium without magnesium supplementation; anddosage is the daily amount of magnesium taken.

The bioavailability comparison of various magnesium compounds utilizingthis methodology were determined in several human subjects. We collecteddata for magnesium gluconate+milk, magnesium diglycinate and magnesiumgluconate. Results are shown in FIG. 25. For comparison, theavailability of other magnesium compounds determined by others is alsoshown in FIG. 25. See Muhlbauer, et al., Eur. J. Clin. Pharmacol.,40:437-8 (1991); see also Bohmer, et al., Magnes. Trace Elem. 9: 272-8(1990). This study demonstrates that there are differences inbioavailability among magnesium paired with different counter ions andthat, for some counter ions, delivery of magnesium with milk enhancesbioavailability.

Example 24 Measuring Plasma, Serum or Urine Magnesium Concentration

Two groups of 6 AD mice were each fed an magnesium diet (test group) anda normal diet (control group) at 5 month of age, respectively, asdescribed above. The cognitive function of the two groups of animals wasthen assessed at 21 mouth of age using the novel object recognition testas described above. After the test, the animals were anesthetized with10% chloral hydrate (4 ul per gram) and then transcardially perfusedwith ice-cold PBS (pH 7.4, without CaCl₂ and MgCl₂) and 4%paraformaldehyde. Next, the whole brain of each animal was immediatelyremoved and post-fixed in 4% paraformaldehyde at 4° C. for 2 hours atroom temperature. The brainstem portion was cut off the whole brain in aclean dish cover and then placed in a 15 ml-sized tube to measure theweight of the tissue. Eight mL concentrated nitric acid was added toeach tupe containing tissue. The tubes were then placed in a sampledigestion microwave oven to digest the samples using a programmedthree-stage digestion procedure according to the table 1

TABLE 1 Microwave digestion steps Heating Ultimate time Pressuretemperature Holding time Step Power (W) (min) (Psi) (° C.) (min) 1 12006 800 120 2 2 1200 3 800 150 2 3 1200 5 800 180 20

The pellucid solutions formed after the digestion were cooled to roomtemperature and then each transferred to a separate beaker with NanoPurewater. The nitric acid in the beakers was removed by evaporation at 170°C. The residue in each beaker was then re-diluted to 25 ml in avolumetric flask. The magnesium contents of the solutions weredetermined by inductively coupled plasma optical emission spectroscopy(ICP-OES). (IRIS, Intrepid II XSP, Thermo Electron, USA). From the totalamount of the magnesium in each solution and the weight of the tissuesample, the magnesium concentration of the brainstem was obtained.

Correlation between brain magnesium concentration and daily magnesiumintake or between cognitive function level and brain magnesiumconcentration was plotted and is shown in FIG. 26. Panel A demonstratesthe correlation between magnesium concentration in the brain (mgmagnesium per gram tissue) and the amount of magnesium daily intake (mgmagnesium per gram body weight). Panel B demonstrates the correlationbetween short-term memory (as assessed by the novel recognition test)and magnesium concentration in the brain. As can be seen from theseresults, we have found that the amount of magnesium intake in AD mice islinearly correlated to the amount of brain magnesium, which in turn waslinearly correlated to the level of cognitive function. This datastrongly suggests a causal relationship between elevation of brainmagnesium level and improvement of cognitive function.

Example 25 Measuring Plasma, Serum or Urine Magnesium Concentration

Another way to define the bioavailability of a magnesium composition isthe ability of the composition to deliver magnesium to tissues. In manyways, this is the ultimate criteria for judging the bioavailability of amagnesium composition. Merely to deliver magnesium to the blood streamis no guarantee that the magnesium will enter the right tissues becausethe newly absorbed magnesium may simply excreted from the urine. Asshown in the previous example, for improved cognitive function, it isimportant that magnesium be delivered to the brain.

Magnesium threonate is better in targeting magnesium to the brain,compared with magnesium gluconate in milk as shown in FIG. 27A. This isa surprising finding as other studies indicate that magnesium gluconatein milk has higher bioavailability to the blood than magnesium threonate(data not shown). Animal behavior data also supports that magnesiumthreonate is better than magnesium gluconate in milk at deliveringmagnesium to the brain. FIG. 27B shows that rats receiving magnesiumthreonate supplements in water (as described previously) at theindicated amount showed marked improvement in their short term memory ina novel object recognition test (as described previously). FIG. 27Cshows that rats receiving magnesium gluconate dissolved in milk did notdemonstrate any improvement in short term memory function in anovel-object recognition test.

These data indicate that the effectiveness of raising brain magnesium bya given magnesium compound is desirable enhancing the animals' memoryfunction. Furthermore, the data suggest that the threonate counter ionmay facilitate the absorption of magnesium by tissues, particularlybrain tissues. Thus, in addition to the use of magnesium threonate forsupplementing magnesium, differential utilization of magnesium-counterion compositions may yield a variety of other possible methods forincreasing magnesium absorption by targeted tissues. For example, anon-magnesium threonate may be used in combination with any othersuitable magnesium compound for enhanced bioavailability of thecompound. Examples of non-magnesium threonate compounds include, but arenot limited to, sodium threonate, potassium threonate, threonic acid,calcium threonate. Alternatively, a precursor threonate compound may beused in the same manner. Examples of such a precursor threonate compoundinclude but not limited to ascorbate and a threonate ester. Ascorbate ismetabolized in the body to form threonate, while a threonate ester, suchas threonate ethyl ester can become hydrolyzed in the body to formthreonate. When a threonate or a precursor threonate compound is used toenhance the bioavailability of another magnesium compound, the twocompounds may or may not be physically combined. When taken separately,they may be taken at the same time or taken at separate times.

Example 26 Measuring Magnesium Concentration Under Fasting Conditions toDetermine Supplement Levels

This example provides one method of the present invention developed toincrease [Mg]_(o), the concentration of Mg²⁺ in the extracellularcompartment, to a predetermined target level. This change of [Mg]_(o)achieves an improvement of various physiological functions.

Unlike for sodium or calcium, there do not appear to be major hormonalhomeostatic mechanisms for regulating serum magnesium. The normal rangeis the result of a balance between the gastrointestinal and renalabsorption and the excretion processes. For this purpose, we analyze thein- and out-flux of magnesium in a multi-compartment model. Thedescription of the multi-compartment model is given next:

Mg_(f) is the amount of magnesium absorbed through food each day,[Mg]_(o) is the concentration of Mg²⁺ in the extracellular compartment,[Mg]_(i) is the concentration of Mg²⁺ in the intracellular compartment,Mg_(u) is the daily excretion of Mg from the kidney, Mg_(s) is the dailyloss of magnesium through sweat, and k_(+i) and k_(−i) are the rateconstants of the Mg²⁺ governing the exchange between [Mg]_(o) and[Mg]_(i). Under the equilibrium condition, net flux (all represented bythe total amount for one day) from [Mg]_(o) to [Mg]_(i) are zero, i.e.inflow and outflow perfectly balance:

Mg _(f) =Mg _(u)([Mg] _(o) ¹)+Mg _(s).  (1)

Next, we describe the case, where one decides to increase [Mg]_(o) ¹ tothe higher value [Mg]_(o) ². To achieve this goal, one needs in theequilibrium to take exactly enough absorbed supplement Mg_(su) to coverthe additional loses

Mg _(f) +Mg _(su) =Mg _(u)([Mg] _(o) ²)+Mg _(s),  (2)

where Mg_(u)([Mg]_(o) ²) is the Mg in urine after the Mg supplement hasbeen added and the new equilibrium has been reached. If we rearrange theequation, we get Mg_(f)−Mg_(s)+Mg_(su)=Mg_(u)([Mg]_(o) ²) andMg_(f)−Mg_(s)=Mg_(u)([Mg]_(o) ¹). This leads to

Mg _(su) =Mg _(u)([Mg] _(o) ²)−Mg _(u)([Mg] _(o) ¹).  (3)

To calculate the Mg_(su) required to achieve [Mg]_(o) ², one needs todetermine the relationship between [Mg]_(o) and Mg_(u). Relationshipbetween [Mg]_(o) and Mg_(u)

In the kidney, Mg in blood is filtered by glomerulus and reabsorbed intubular cells. The amount of Mg filtered is the products of theglomerular filtration rate (GFR), [Mg]_(o), and the molecular weight ofMg (Mg_(mw)) (GFR·[Mg]_(o)·Mg_(mw)). The filtered magnesium isreabsorbed in renal tubules. When [Mg]_(o) is below a certain point, thekidney is capable of retaining all of the filtered Mg, and Mg_(u) isnear zero. At this point, the urine magnesium excretion seems linearlycorrelated with [Mg]_(o). To quantify this process, we studied therelationship between [Mg]_(o) and Mg_(u) in 3 human volunteers. Theblood and urine magnesium were sampled every four hours in day duringfasting. Their relationships are plotted in FIG. 28A. Evidently, therelationship between urine magnesium and [Mg]_(o) is linear.

From this data, one can get an empirical formula that predicts thegeneral relationship between [Mg]_(o) and Mg_(u) in the relevant dailyphysiological range of 0.7-0.85 mM, i.e. range achieved withoutextensive fasting. We define [Mg]_(o) at the point where urine losses goto zero to be [Mg]_(basal). The excretion of Mg through kidney mightthen be taken to be proportional to [Mg]_(o)−[Mg]_(basal). Thus, for agiven GFR and a period of time (T (hour)), we get

$\begin{matrix}{\frac{{Mg}_{u}\left( \lbrack{Mg}\rbrack_{o} \right)}{{GFR}*T_{s}} = {{Mg}_{mw}*k_{s}*\left( {\lbrack{Mg}\rbrack_{o} - \lbrack{Mg}\rbrack_{basal}} \right)}} & (4)\end{matrix}$

Where k_(e) is the proportionality constant, which physiologicallydefines the rate of Mg loss through the kidneys at a given [Mg]_(o). Thedata fitting with equation 4 seems sufficient to predict therelationship between [Mg]_(o) and [Mg]_(u) (FIG. 28A).

Combining equation 3 and 4, the amount of net Mg needed as a supplementto achieve a higher [Mg]_(o) can be predicted by the following equation:

Mg _(su) =GFR·T·Mg _(mw) ·k _(e)·([Mg] _(o) ² −[Mg] _(o) ¹)  (5)

For a Mg compound X with bioavailability of k_(x), the amount of Mgcompound one needs to take is Mg_(X)=Mg_(su)/k_(x).Applying the above to Routine followed by users to determine initial Mgstatus, choice of correct supplement amount and feedback loop to achievedesired result:

1) Determine body Mg status: using [Mg]_(plasma) at 9:00 AM beforebreakfast and after fasting 12 hours.

2) Decide the target [Mg]_(plasma)

3) Calculation of k_(e) and [Mg]_(basal) using following procedures:

-   -   a. Day one: Measure [Mg]_(plasma) at 9:00 AM before breakfast        and collect Mg_(u) from 8:30 AM to 10:30 AM.    -   b. Measure [Mg]_(plasma) at 3:00 PM and collect Mg_(u) from 2:30        PM to 4:30 PM (2-4 hours after lunch at the expected peak of        [Mg]_(plasma) and Mg_(u)).    -   c. Day two: Take 300 mg magnesium Gluconate dissolved in 200 ml        of milk at 12:00 PM with normal food. Measure [Mg]_(plasma) at        3:00 PM and collect Mg_(u) from 2:30 PM to 4:30 PM.    -   d. From the blood and urine sample, one can determine averaged        GFR for each pair of blood and urine samples.    -   e. Plot the collected data and fit them with a linear equation

$\frac{{Mg}_{u}\left( \lbrack{Mg}\rbrack_{o} \right)}{{GFR}*T_{s}} = {{{Mg}_{mw}*k_{e}*\lbrack{Mg}\rbrack_{plasma}} + b}$

-   -   f. Finally,

[Mg] _(basal) =−b/(Mg _(mw) ·k _(e))  (6)

-   -   g. See FIG. 28B

4) Optimal Dosage:

With the parameters determined from above procedures, one can calculatethe proper dosage with following equations.

Mg _(x) =GFR·T·Mg _(mw) ·k _(e)·([Mg] _(o) ² −[Mg] _(o) ¹)/k _(x)  (7)

Predictions for three human subjects utilizing this method are shown inTable 2.

Subj. GFR Time [Mg]basal [Mg]initial [Mg]final ke U initial U final MgsuKx MgX L 7.5 24 0.67 0.78 0.88 0.19 93 175 82 0.3 273 Z 7.5 24 0.69 0.780.88 0.28 112 233 122 0.3 405 LX 7.5 24 0.72 0.77 0.88 0.51 118 364 2460.3 820

5) The most effective way of loading: A sustained-release form of Mgcompound (within 12 hours) taken before sleep.

6) checking procedures:

-   -   a. Previous study suggests that 6 to 18 days are required for        equilibrium to be established following changes in magnesium        intake. We recommend checking body Mg status 1 month after daily        Mg supplement intake has started, assuming that Mg status has        already reached approximately the new equilibrium. The        [Mg]_(plasma) and urine Mg will be taken using same procedure        listed in step 3a without taking Mg supplement in day before        testing. If the dosage is appropriate, [Mg]_(plasma) will be        close (+/−10%, more accurately +5% to −15% of the correct value,        since the approach is from below) to the desired level and        Mg_(u) will be close to

Mg _(U) =GFR·T·Mg _(mw) ·k _(e)·([Mg] _(o) ² −[Mg] _(basel))

-   -   b. If [Mg]_(plasma) and Mg_(u) deviate from the target values,        the error is most likely due to an inaccurate estimate of k_(x).        As bioavailability (k_(x)) for a Mg compound might not be        constant among the population, one can use the these data to        calculate the efficacy of loading Mg compound into intracellular        compartment (k′_(x)).

k _(x)′=(Mg _(u) ² −Mg _(u) ¹)/Mg _(x)  (8)

-   -   -   When k′_(x) is determined, equation 7 can be used to            recalculate the dosage and check the [Mg]_(plasma) and            Mg_(u) one month later. This procedure can be repeated until            the [Mg]_(plasma) reaches the desired value.

    -   c. Procedure 6b is preferably repeated biannually.

Example 27 Effect of Magnesium Treatment on Synaptic Protection in ADMice

In this example we examine the ability of magnesium threonate treatmentto protect against synapse loss in AD mice. The same group of animalsused for the memory test in example 14 are sacrificed. The brains of theanimals were then fixed for electronmicroscopic analysis to count thenumber of synapses per unit area (synaptic density). Samples werestained so as to indicate the synapses (FIGS. 29 A and B, synapsesindicated by arrows).

FIG. 29A shows the lower synapse count in the dentate gyrus of thehippocampus of AD mice. FIG. 29B shows the higher synaptic density inthe same region in AD mice treated with magnesium threonate supplementeddiet. FIG. 29C shows the results of a quantitative comparison of thesynaptic densities in AD mice, AD mice receiving magnesium threonatetreatment, and wild type mice. The synaptic density in AD mice issignificantly lower tan for the wild type mice or AD mice under MgTtreatment (p<0.001). However, the synaptic density in AD mice receivingmagnesium threonate treatment is more similar to wild type mice. Theseresults indicate the protective effect of magnesium treatment onsynaptic loss in AD progression.

A composition for administration to a subject, such as oraladministration to a subject, for example, has been described herein.Such a composition may comprise at least one magnesium-counter ioncompound. A magnesium-counter ion composition described herein may beuseful for any of a variety of applications and purposes describedherein, such as maintaining, enhancing, and/or improving health,nutrition, and/or another condition of a subject, and/or cognitive,learning, and/or memory function, for example. A magnesium-counter ioncomposition described herein may be useful for administration to asubject presenting magnesium deficiency, mild cognitive impairment,Alzheimer's disease, attention deficit hyperactivity disorder, ALS,Parkinson's disease, diabetes, migraine, anxiety disorder, mooddisorder, and/or hypertension, merely by way of example.

A kit may comprise at least one component of any magnesium-counter ioncomposition described herein or any magnesium-counter ion compositiondescribed herein. A kit may further comprise a vehicle for administeringat least one such component or such a composition to a subject, such asa drinking vessel for a liquid component or composition, merely by wayof example, or a holding vessel for any component or composition and avehicle for moving same from the holding vessel to a mouth of a subject,such as a bowl and a spoon, merely by way of example.

A method of providing magnesium supplementation to a subject may beuseful to a subject in any of the ways described herein. Such a methodmay comprise administering to a subject, such as orally administering toa subject, at least one magnesium-counter ion compound. Such a methodmay comprise providing any suitable amount, concentration, or a dosageof elemental magnesium associated with the at least onemagnesium-counter ion compound to a subject.

A composition and/or a method described herein may be useful forpurposes described herein, such as maintaining, enhancing, and/orimproving health, nutrition, and/or another condition of a subject,and/or cognitive, learning, and/or memory function, for example, such asmagnesium deficiency, mild cognitive impairment, Alzheimer's disease,attention deficit hyperactivity disorder, ALS, Parkinson's disease,diabetes, migraine, anxiety, mood, and hypertension, merely by way ofexample. A composition and/or a method described herein may be usefulfor administration to a subject presenting magnesium deficiency, mildcognitive impairment, Alzheimer's disease, attention deficithyperactivity disorder, ALS, Parkinson's disease, diabetes, migraine,anxiety disorder, mood disorder, and/or hypertension, merely by way ofexample.

Various modifications, processes, as well as numerous structures thatmay be applicable herein will be apparent. Various aspects, features orembodiments may have been explained or described in relation tounderstandings, beliefs, theories, underlying assumptions, and/orworking or prophetic examples, although it will be understood that anyparticular understanding, belief theory, underlying assumption, and/orworking or prophetic example is not limiting. Although the variousaspects and features may have been described with respect to variousembodiments and specific examples herein, it will be understood that anyof same is not limiting with respect to the full scope of the appendedclaims or other claims that may be associated with this application.

The examples set forth above are given to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use various embodiments of the methods and systems disclosedherein, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of enhancing cognitive function comprising administering toa subject an amount of magnesium-containing compound effective toachieve a physiological concentration of magnesium at about 0.75 mM orabove, wherein said concentration of magnesium is measured under afasting condition.
 2. The method of claim 1, wherein said concentrationof magnesium is measured after fasting for at least about twelve hours.3. The method of claim 1, wherein said physiological concentration isserum concentration, plasma concentration, or cerebrospinal fluidconcentration.
 4. The method of claim 1, wherein said physiologicalconcentration is cerebrospinal fluid concentration.
 5. The method ofclaim 1, wherein said magnesium-containing compound is amagnesium-counter ion compound.
 6. The method of claim 5, wherein saidcounter ion is an organic counter ion.
 7. The method of claim 6, whereinsaid organic counter ion is threonate.
 8. The method of claim 1, whereinsaid magnesium-containing compound is a magnesium-supplementedfoodstuff.
 9. The method of claim 1, wherein said cognitive function isshort-term memory or long-term memory.
 10. The method of claim 1,wherein said concentration is maintained for a period of greater than 1month.
 11. A method of maintaining cognitive function comprisingadministering to a subject an amount of magnesium-containing compoundeffective to increase a physiological concentration of magnesium by atleast about 10% as compared to an initial level of magnesium prior tosaid administration.
 12. The method of claim 11, wherein said increaseis measured under a fasting condition.
 13. The method of claim 11,wherein said physiological concentration is serum concentration, plasmaconcentration, or cerebrospinal fluid concentration.
 14. The method ofclaim 11, wherein said magnesium-containing compound is amagnesium-counter ion compound.
 15. The method of claim 14, wherein saidcounter ion is an organic counter ion.
 16. The method of claim 15,wherein said organic counter ion is threonate.
 17. The method of claim15, wherein said magnesium-containing compound is amagnesium-supplemented foodstuff.
 18. The method of claim 11, whereinsaid concentration is maintained for a period of greater than 4 months.19. The method of claim 11, further comprising the step of determiningstarting physiological magnesium concentration of said subject under afasting condition.
 20. A method of maintaining and/or enhancingcognitive function comprising administering to a subject an amount ofmetal-organic counter ion complex effective to increase a physiologicalconcentration of threonate by at least about 10% as compared to aninitial level of threonate prior to said administration.
 21. The methodof claim 20, wherein said metal-organic counter ion complex comprisesthreonate as a counter-ion.
 22. A method of therapeutic or prophylactictreatment of cognitive dysfunction, comprising administering to asubject in need of therapeutic or prophylactic treatment of cognitivedysfunction a magnesium-containing composition to yield a level ofphysiological concentration of magnesium sustained at the level of 0.75mM or above for at least about 15 days.
 23. The method of claim 22,wherein magnesium is sustained at the level of 0.75 mM or above for atleast about 1 month.
 24. The method of claim 22, wherein magnesium issustained at the level of 0.75 mM or above for at least about 4 months.25. The method of claim 22, wherein magnesium concentration is magnesiumplasma concentration measured after fasting for at least about 8 hours.26. The method of claim 1 wherein the subject is an adult.
 27. Themethod of claim 1 wherein the subject is a patient suffering from ordiagnosed with dementia.
 28. The method of claim 1 wherein the subjectis a patient suffering from or diagnosed with Alzheimer's disease.